Test strip for lateral flow chromatography, analyte measuring method and analyte measuring kit

Abstract

To quantify an analyte with high accuracy, in immunoassay.SOLUTION: A test strip 100 is provided with a determination unit 20 and a control unit 30, arranged in this order in a direction of sample development indicated by an arrow. The test strip 100 comprises a sample addition unit 40 and a reaction unit 50 upstream of the determination unit 20, and a liquid absorption unit 60 downstream of the control unit 30. The control unit 30 has a plurality of control lines CL1, CL2. By providing the plurality of control lines, it is possible to capture preferably 90% or more of migrating marker antibodies in the determination unit 20 and the control unit 30.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a lateral flow chromatographic test strip used in immunoassays, an analyte measurement method, and an analyte measurement kit. [Background technology]

[0002] Immunoassay is a method for qualitatively and quantitatively analyzing trace components by utilizing specific reactions between antigens and antibodies. In immunoassays, detection sensitivity is increased by binding a labeling substance to an antibody, an antigen, or a complex thereof. Therefore, the labeling ability of a labeling substance can be said to be an important factor that determines the detection ability in immunoassays. It has been proposed to use metal-resin composite particles, which are composites of metal particles and resin particles, as labeling substances for immunoassays (e.g., Patent Documents 1 to 3).

[0003] Proposals have also been made to enable quantification of analytes by immunoassay (e.g., Patent Document 4). Patent Document 4 discloses a reverse lateral flow immunoassay method designed to detect human antigen-specific immunoglobulin E (IgE). However, it cannot be said that a sufficient correlation has been obtained between the concentration of the analyte in the sample and the color intensity, and it is considered insufficient for highly accurate quantification of the analyte. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication WO2016 / 002742 [Patent Document 2] International Publication WO2017 / 010391 [Patent Document 3] International Publication WO2018 / 123952 [Patent Document 4] Special Publication No. 2023-506679 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to quantify an analyte with high accuracy in an immunoassay. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors have discovered that the above problems can be solved by providing a plurality of control lines in the control section of a chromatography test strip, and have thus completed the present invention.

[0007] That is, the lateral flow chromatography test strip of the present invention is a lateral flow chromatography test strip for detecting or quantifying an analyte contained in a sample. The lateral flow chromatography test strip of the present invention comprises a determination part having a first capture ligand immobilized thereon that specifically binds to the analyte; a control section, located downstream of the determination section in the direction of sample development, in which a second capture ligand that specifically binds to a labeled antibody obtained by labeling an antibody that specifically binds to the analyte with a labeling substance is immobilized; It is equipped with: In the lateral flow chromatography test strip of the present invention, the control section has a plurality of control lines spaced apart in the direction in which the sample develops.

[0008] The lateral flow chromatography test strip of the present invention may have two to five control lines.

[0009] In the lateral flow chromatography test strip of the present invention, the amount of the second capture ligand immobilized on the control lines may be equal to or different from each other.

[0010] When the lateral flow chromatography test strip of the present invention has three or more control lines, the control lines may be arranged at equal intervals or at different intervals in the direction in which the sample develops.

[0011] The lateral flow chromatography test strip of the present invention may further include a reaction zone containing the labeled antibody upstream of the determination zone, The amount Q2 that can be captured by the second capture ligand relative to the initial total amount Q1 of the labeled antibody in the reaction zone is 0.9×Q1≦Q2≦1.0×Q1 There may be a relationship between the above.

[0012] In the lateral flow chromatography test strip of the present invention, the labeled substance may be a labeled substance comprising a metal-resin complex having a resin particle and a plurality of metal particles immobilized on the resin particle.

[0013] The method for measuring an analyte of the present invention is a method for detecting or quantifying an analyte contained in a sample using a lateral flow chromatographic test strip, and uses the above-mentioned lateral flow chromatographic test strip of the present invention. The method for measuring an analyte of the present invention comprises the following steps (I) to (V): Step (I): contacting the sample with a labeled antibody obtained by labeling an antibody that specifically binds to the analyte with a labeling substance; Step (II): contacting a conjugate of the analyte and the labeled antibody with the first capture ligand in the determination section; Step (III): contacting the labeled antibody and / or the conjugate with the second capture ligand in the control section; Step (IV): measuring the color intensity caused by the labeling substance in the judgment section and the control section, respectively; Step (V): quantitating the amount of analyte contained in the sample based on the measured color intensity; Contains:

[0014] In the step (V) of the method for measuring an analyte of the present invention, the color intensity in the judgment zone may be corrected by dividing it by the sum of the color intensities in the judgment zone and the control zone, and then the amount of analyte contained in the sample may be estimated.

[0015] In the method for measuring an analyte of the present invention, the sum of the amount of labeled antibody in the conjugate captured by the first capture ligand in step (II) and the amount of labeled antibody captured by the second capture ligand in step (III) may be 90% or more of the initial total amount of labeled antibody used in step (I).

[0016] The analyte measurement kit of the present invention is a kit for detecting or quantifying an analyte contained in a sample, and includes the above-mentioned lateral flow chromatography test strip of the present invention.

[0017] The analyte measurement kit of the present invention may further comprise reference standard data showing the relationship between the color intensity or a corrected value thereof in the determination zone and the concentration of the analyte. [Effects of the Invention]

[0018] The lateral flow chromatography test strip of the present invention has multiple control lines in the control section, which enables a sufficient correlation to be obtained between the analyte concentration and color intensity in immunoassays, with little variation in measurement results, enabling highly sensitive detection of analytes in samples. Therefore, by using the lateral flow chromatographic test strip of the present invention, highly sensitive detection and highly accurate quantification of analytes become possible, and the test strip can be preferably applied to various immunological measurements. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is an explanatory diagram showing an overview of a lateral flow chromatography test strip according to one embodiment of the present invention. [Figure 2] FIG. 1 is a cross-sectional view of a test strip used in a test example. DETAILED DESCRIPTION OF THE INVENTION

[0020] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0021] [Lateral flow chromatography test strips] First, a lateral flow chromatography test strip (hereinafter, simply referred to as a "test strip") according to one embodiment of the present invention will be described with reference to Figure 1. This test strip 100 can be preferably used in the analyte measurement method according to one embodiment of the present invention, as will be described later.

[0022] The test strip 100 includes a membrane 10. The test strip 100 includes, in order, a determination section 20 and a control section 30 in the direction of sample development indicated by the arrow in Fig. 1. The test strip 100 also includes a sample addition section 40 and a reaction section 50 upstream of the determination section 20 in the direction of sample development, and a liquid-absorbing section 60 downstream of the control section 30.

[0023] <Membrane> The membrane 10 used in the test strip 100 can be made of a material commonly used as a membrane in immunoassays. The membrane 10 is formed of a microporous and inert material (a material that does not react with analytes, various ligands, etc.) that exhibits capillary action and, upon addition of a sample, develops in the direction indicated by the arrow in FIG. 1. Specific examples of the membrane 10 include fibrous or nonwoven fibrous matrices made of polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, cellulose derivatives, etc., membranes, filter paper, glass fiber filter paper, cloth, cotton, etc. Among these, membranes, filter paper, glass fiber filter paper, etc. made of cellulose derivatives or nylon are preferred, and nitrocellulose membranes, mixed nitrocellulose ester (a mixture of nitrocellulose and cellulose acetate) membranes, nylon membranes, and filter paper are more preferred.

[0024] For easier operation, the test strip 100 preferably includes a support 70 for supporting the membrane 10. The support 70 may be, for example, a plastic case or sheet.

[0025] <Judgment part> A first capture ligand that specifically binds to the analyte is immobilized on the determination section 20. The immobilized first capture ligand forms a test line. The test line is not limited to one, and may be two or more. The first capture ligand can be any ligand that specifically binds to the analyte, and for example, an antibody against the analyte is preferably used. The first capture ligand is immobilized so that it does not move from the determination section 20 even when a sample is provided on the test strip 100. The first capture ligand may be directly or indirectly immobilized on the membrane 10 by physical or chemical bonding, adsorption, or the like.

[0026] The determination unit 20 is not particularly limited as long as it has a configuration that allows a conjugate of an analyte and a labeled antibody (hereinafter sometimes referred to as an "analyte-labeled antibody conjugate") to come into contact with a first capture ligand that specifically binds to the analyte. For example, the first capture ligand may be immobilized directly on the membrane 10, or the first capture ligand may be immobilized on a pad made of cellulose filter paper, glass fiber, nonwoven fabric, or the like that is fixed to the membrane 10.

[0027] <Control section> The test strip 100 has a control section 30 formed on the membrane 10 downstream of the determination section 20 in the direction of sample development. The control section 30 has a second capture ligand immobilized thereon that specifically binds to the labeled antibody. The immobilized second capture ligand forms a control line. By measuring the color intensity in the control section 30, it can be confirmed that the sample applied to the test strip 100 has developed and reached the determination section 20, and that the test has been performed normally.

[0028] In the present invention, the control section 30 is provided with a plurality of control lines spaced apart in the direction of sample development. The test strip 100 shown in FIG. 1 has two control lines CL1 and CL2. By providing a plurality of control lines, it becomes possible to capture almost all of the labeled antibody and analyte-labeled antibody conjugate that migrate through the test strip 100 in the determination section 20 and the control section 30. More specifically, it becomes possible to capture the labeled antibody and analyte-labeled antibody conjugate with a high capture yield of preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more of the total amount of the migrated labeled antibody and analyte-labeled antibody conjugate.

[0029] In the prior art, the control section had no function other than to confirm whether the sample had developed and reached the determination section 20. Therefore, if the developing labeled antibody could not be captured by a single control line, the color intensity became saturated, and the color intensity at the control line could not be used for quantitative measurement. In contrast, in the present invention, by providing multiple control lines, the developed labeled antibodies (including analyte-labeled antibody conjugates) can be captured with a high capture yield, nearly to the entire amount, in the determination section 20 and the control section 30. In other words, the labeled antibodies (including analyte-labeled antibody conjugates) that were not captured by the control line CL1 can be captured by the downstream control line CL2. As a result, it becomes possible to accurately grasp the relationship between the color intensity of the analyte-labeled antibody conjugate captured by the test line and the color intensity based on the total amount of labeled antibodies developed on the test strip 100, and by performing correction based on the calculation formula (1) or (2) described below, it becomes possible to quantify the analyte with high accuracy.

[0030] The number of control lines is not limited to two and may be more than two. For example, the number of control lines is preferably in the range of 2 to 5, more preferably in the range of 2 to 4, and most preferably 2 or 3.

[0031] The amounts of the second capture ligand immobilized on the control lines CL1 and CL2 may be equal or different. When the amounts of the second capture ligand immobilized on the control lines CL1 and CL2 are different, the amount of the second capture ligand on the upstream control line CL1 in the sample development direction may be greater or less than the amount of the second capture ligand on the downstream control line CL2. Preferably, the amount of the second capture ligand on the control line CL2 is greater than that on the control line CL1, thereby enabling the capture of more particles that were not captured by the control line CL1. Furthermore, when adjusting the amounts of the capture ligand on the control lines CL1 and CL2, it is preferable to adjust the amounts of the capture ligand so that the color intensity of the control lines CL1 and CL2 measured by a measuring device such as an immunochromatography reader falls within a quantitatively measurable range according to the specifications of the measuring device.

[0032] Furthermore, when there are three or more control lines, the control lines may be arranged at equal intervals in the direction of sample development, or may be arranged at different intervals.

[0033] Furthermore, the amount Q2 that can be captured by the second capture ligand in the control section 30 relative to the initial total amount Q1 of the labeled antibody in the reaction section 50 is 0.9×Q1 ≦Q2 ≦1.0×Q1 Here, the captureable amount Q2 means the total captureable amount of the multiple control lines CL1 and CL2.

[0034] The control section 30 is not particularly limited as long as it has a configuration that allows the labeled antibody or analyte-labeled antibody conjugate to come into contact with a second capture ligand that specifically binds to the labeled antibody. For example, the second capture ligand may be immobilized directly on the membrane 10, or the second capture ligand may be immobilized on a pad made of cellulose filter paper, glass fiber, nonwoven fabric, or the like that is fixed to the membrane 10.

[0035] <Sample addition section> The test strip 100 may have a sample application section 40 for applying a sample containing an analyte. The sample application section 40 is a site for receiving the sample containing an analyte in the test strip 100. The sample application section 40 may be formed on the membrane 10 upstream of the determination section 20 in the direction in which the sample develops, or the sample application section 40 may be formed by fixing a sample application pad made of a material such as cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, or cotton cloth to the membrane 10.

[0036] <Reaction section> The test strip 100 may have a reaction unit 50 containing a labeled antibody formed on the membrane 10. The reaction unit 50 may be located upstream of the determination unit 20 in the direction of sample flow. The sample addition unit 40 in FIG. 1 may also be used as the reaction unit 50. When the test strip 100 has the reaction unit 50, when a sample containing an analyte is supplied to the reaction unit 50 or the sample addition unit 40, the analyte contained in the sample can be brought into contact with the labeled antibody in the reaction unit 50. In this case, simply supplying the sample to the reaction unit 50 or the sample addition unit 40 can form an analyte-labeled antibody conjugate, thereby enabling so-called one-step immunochromatography.

[0037] The reaction unit 50 is not particularly limited as long as it contains a labeled antibody that specifically binds to the analyte, but may be one in which the labeled antibody is directly applied to the membrane 10. Alternatively, the reaction unit 50 may be one in which a pad (conjugate pad) made of, for example, cellulose filter paper, glass fiber, nonwoven fabric, or the like, impregnated with the labeled antibody is immobilized on the membrane 10.

[0038] <Liquid suction part> The liquid-absorbent part 60 is formed by a pad of a water-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 liquid-absorbent part 60 varies depending on the material and size of the liquid-absorbent part 60. Therefore, by selecting the material, size, etc. of the liquid-absorbent part 60, it is possible to set the development speed optimal for the detection and quantification of analytes. The liquid-absorbent part 60 is an optional component and may be omitted.

[0039] Test strip 100 may further include any desired portion or member, as needed.

[0040] [Analyte measurement method] Next, a method for measuring an analyte according to one embodiment of the present invention, which is carried out using the test strip 100, will be described.

[0041] The method for measuring an analyte of the present invention is a method for detecting or quantifying an analyte contained in a sample, and is carried out using a test strip 100. The method for measuring an analyte of the present invention comprises the following steps (I) to (V); Step (I): contacting a sample with a labeled antibody obtained by labeling an antibody that specifically binds to an analyte with a labeling substance; Step (II): A step of contacting the analyte-labeled antibody conjugate with the first capture ligand in the determination unit 20; Step (III): contacting the labeled antibody and / or analyte-labeled antibody conjugate with a second capture ligand in the control section 30; Step (IV): measuring the color intensity caused by the labeling substance in the determination section 20 and the control section 30, respectively; Step (V): quantitating the amount of analyte contained in the sample based on the measured color intensity; Contains:

[0042] Process (I): Step (I) is a step of contacting a sample with a labeled antibody, which is an antibody that specifically binds to an analyte and is labeled with a labeling substance. In step (I), the manner of contacting the sample with the labeled antibody is not particularly limited, as long as an analyte-labeled antibody conjugate can be formed. For example, the sample may be provided to the sample application section 40 or the reaction section 50 of the test strip 100, and the analyte may be contacted with the labeled antibody in the sample application section 40 or the reaction section 50. Alternatively, the analyte in the sample may be contacted with the labeled antibody before the sample is provided to the test strip 100. If the analyte is not present in the sample, an analyte-labeled antibody conjugate will not be formed.

[0043] The analyte-labeled antibody complex formed in step (I) develops and migrates on the test strip 100 to reach the test zone 20 .

[0044] Process (II): In step (II), the analyte-labeled antibody conjugate is brought into contact with a first capture ligand in the test strip 100's test zone 20. When the analyte-labeled antibody conjugate is brought into contact with the first capture ligand, the analyte in the analyte-labeled antibody conjugate specifically binds to the first capture ligand. As a result, the analyte-labeled antibody conjugate is captured in the test zone 20.

[0045] Since the first capture ligand does not specifically bind to the labeled antibody, when the labeled antibody that is not bound to the analyte reaches the determination zone 20, the unbound labeled antibody passes through the determination zone 20.

[0046] Process (III): Step (III) is a step of contacting the labeled antibody and / or analyte-labeled antibody conjugate with a second capture ligand in the control section 30. The labeled antibody that is not bound to the analyte continues to develop as it passes through the determination section 20, and binds to the second capture ligand in the control section 30. As a result, the labeled antibody that is not bound to the analyte is captured in the control section 30. Furthermore, if the amount of analyte in the sample is greater than the amount that can be captured by the test line in the determination section 20, the analyte-labeled antibody conjugate passes through the determination section 20 and continues to develop, and in the control section 30 it binds to the second capture ligand at the specific binding site of the labeled antibody and is captured.

[0047] After step (III), if necessary, and before step (IV), a washing step may be performed in which the test strip 100 is washed with a buffer solution commonly used in biochemical tests, such as water, physiological saline, or phosphate buffer solution. The washing step can remove labeled antibodies that have not been captured in the determination zone 20 and the control zone 30. By performing the washing step, the background color intensity can be reduced when measuring the color intensity in the determination zone 20 and the control zone 30 in step (IV), thereby increasing the signal-to-background ratio and further improving detection sensitivity and quantitation.

[0048] Process (IV): Step (IV) is a step of measuring the color intensity caused by the labeling substance in the determination section 20 and the control section 30. After performing step (III) or a washing step as needed, the color intensity caused by the labeling substance is measured in the determination section 20 of the test strip 100, thereby enabling highly sensitive detection of the analyte contained in the sample. Furthermore, by measuring the color intensity not only in the determination section 20 but also in the control section 30, it is possible to confirm whether the sample provided on the test strip 100 has developed normally and reached the determination section 20.

[0049] The color intensity can be measured using a measuring device such as an immunochromatography reader. The principle of measuring the color intensity in the measuring device is not particularly limited. For example, an immunochromatography reader irradiates the determination unit 20 and the control unit 30 with light and measures the color intensity from the attenuation of the reflected light.

[0050] Step (V) is a step of quantifying the amount of analyte contained in the sample based on the measured color intensity. In step (V), it is preferable to correct the color intensity in the determination zone 20 by dividing it by the sum of the color intensities in the determination zone 20 and the control zone 30, and then estimate the amount of analyte contained in the sample.

[0051] In the present invention, since the control section 30 has a plurality of control lines CL1 and CL2, when the color intensity of the test line in the judgment section 20 is TL, the color intensity of the control line CL1 is CL1, and the color intensity of the control line CL2 is CL2, the following calculation formula (1) is used: TL / (TL+CL1+CL2) … (1) Based on this, correction can be made. If there are n control lines (n is an integer), use the following calculation formula (2): TL / (TL+CL1+CL2+ …CLn) … (2) Correction can be made based on the above. Since the color intensity of the test line and the color intensity of the control line are in a constant proportional relationship, normalization by correction using the above calculation formulas (1) and (2) makes it possible to offset variations in the concentrations of the developed analyte-labeled antibody conjugate and labeled antibody, as well as variations in the development speed, thereby improving quantitation.

[0052] Furthermore, in step (V), reference standard data showing the relationship between the color intensity in the determination unit 20 or the correction value calculated by the above-mentioned formula (2) and the amount of analyte can be used. Examples of reference standard data include a calibration curve, table, or formula showing the relationship between color intensity and analyte concentration. The reference standard data may be prepared for general use, or may be prepared by conducting a preliminary experiment using a standard reagent with a known analyte concentration prior to the immunoassay.

[0053] Furthermore, in the method of the present invention, the sum of the amount of labeled antibody in the analyte-labeled antibody conjugate captured by the first capture ligand in step (II) and the amount of labeled antibody captured by the second capture ligand in step (III) (capture yield) is preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more, relative to the initial total amount of labeled antibody used in step (I). A capture yield of 90% or more makes it possible to accurately grasp the relationship between the color intensity of the analyte-labeled antibody conjugate captured at the test line and the color intensity based on the total amount of labeled antibody developed on test strip 100, and highly accurate quantification is possible by performing correction based on calculation formula (1) or (2).

[0054] <Samples and analytes> The sample used in the analyte measurement method of the present invention is not particularly limited as long as it contains a substance that can serve as an antigen, such as a protein, as the analyte. Examples include biological samples containing the target analyte (i.e., whole blood, serum, plasma, urine, saliva, sputum, nasal or pharyngeal swabs, cerebrospinal fluid, amniotic fluid, nipple secretions, tears, sweat, skin exudates, tissue, cell, and fecal extracts, etc.), food extracts, etc. If necessary, the analyte contained in the sample may be pretreated prior to step (I) above to facilitate the specific binding reaction between the labeled antibody and the first capture ligand and the analyte. Examples of pretreatment include chemical treatment using various chemicals such as acids, bases, and surfactants, and physical treatment using heating, stirring, ultrasound, etc. In particular, when the analyte is a substance that is not normally exposed on a surface, such as influenza virus NP antigen, treatment with a surfactant is preferred. For this purpose, a nonionic surfactant can be used as the surfactant, taking into account the binding reactivity between the first capture ligand and the analyte in specific binding reactions, such as antigen-antibody reactions.

[0055] The sample may also be diluted appropriately with a solvent (water, physiological saline, buffer solution, etc.) or a water-miscible organic solvent used in a typical immunological analysis method.

[0056] The analyte is not particularly limited and can be any known analyte, and can be measured with strong anionic or cationic properties, as well as other analytes. The analyte is not particularly limited as long as it specifically binds to the labeled antibody and the first capture ligand, and examples thereof include tumor markers, signal transduction substances, proteins such as hormones (including polypeptides, oligopeptides, etc.), nucleic acids (including single-stranded or double-stranded DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc.) or substances containing nucleic acids, sugars (including oligosaccharides, polysaccharides, sugar chains, etc.) or substances containing sugar chains, lipids, and other molecules. More specifically, examples of analytes include carcinoembryonic antigen (CEA), HER2 protein, prostate-specific antigen (PSA), CA19-9, α-fetoprotein (AFP), immunosuppressive acidic protein (IAP), CA15-3, CA125, estrogen receptor, progesterone receptor, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), syphilis antibody, influenza virus human hemoglobin, chlamydia antigen, group A beta-hemolytic streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, glycated albumin, etc. Among these, antigens that can be solubilized by nonionic surfactants are preferred, and antigens that form self-aggregates such as viral nucleoproteins are more preferred.

[0057] <Labeled antibody> In step (I), the labeled antibody is contacted with an analyte contained in the sample to form an analyte-labeled antibody conjugate. The labeled antibody is an antibody that specifically binds to an analyte and is labeled with a labeling substance. Here, "labeling" means that in steps (I) to (V), the labeling substance is directly or indirectly immobilized to the antibody by chemical or physical bonding, adsorption, or the like to the extent that the labeling substance is not released from the labeled antibody. For example, the labeled antibody may be one in which the labeling substance is directly bound to the antibody, or one in which the antibody and the labeling substance are bound via an arbitrary linker molecule, or one in which each is immobilized to an insoluble particle.

[0058] Furthermore, in the present invention, the "antibody" is not particularly limited and known antibodies can be used, including those with strong anionic properties, those with strong cationic properties, and other antibodies. For example, polyclonal antibodies, monoclonal antibodies, antibodies obtained by genetic recombination, and antibody fragments capable of binding to antigens [e.g., H chains, L chains, Fab, F(ab')2, etc.] can be used. Furthermore, immunoglobulins may be any of IgG, IgM, IgA, IgE, and IgD. Antibody-producing animal species may include humans as well as non-human animals (e.g., mice, rats, rabbits, goats, horses, etc.). Specific examples of antibodies include anti-PSA antibodies, anti-AFP antibodies, anti-CEA antibodies, anti-adenovirus antibodies, anti-influenza virus antibodies, anti-HCV antibodies, anti-IgG antibodies, and anti-human IgE antibodies.

[0059] The labeling substance is not particularly limited as long as it can be used in immunoassays, and examples that can be used include metal-resin composite particles, gold colloid particles, platinum colloid particles, colored latex particles, fluorescent substance particles, magnetic particles, etc. Among these, metal-resin composite particles formed by combining metal particles and resin particles are preferred because they can improve the accuracy of quantitative measurements, and for example, the metal-resin composite particles described in Patent Documents 1 to 3 are more preferred.

[0060] A specific example of a metal-resin composite particle is a metal-resin composite particle having a resin particle and a plurality of metal particles fixed to the resin particle. In the metal-resin composite particle, the metal particles preferably include metal particles completely encapsulated in the resin particle (encapsulated particles), and metal particles having portions embedded in the resin particle and portions exposed outside the resin particle (partially exposed particles). In addition, metal particles adsorbed to the surface of the resin particle (surface-adsorbed particles) may also be present. Furthermore, it is preferable that at least some of the metal particles are three-dimensionally distributed in the surface layer of the resin particle. Here, the "surface layer" refers to a range of 50% of the particle radius in the depth direction from the surface of the resin particle, based on the outermost position of the metal-resin composite (i.e., the protruding end of the partially exposed particle or surface-adsorbed particle). Furthermore, "three-dimensionally distributed" means that the metal particles are dispersed not only in the plane direction of the resin particle but also in the depth direction. The metal-resin composite particles are preferably made of platinum or an alloy thereof, or gold or an alloy thereof, and preferably have an average particle size in the range of 50 nm to 1000 nm.

[0061] [Analyte measurement kit] An analyte measurement kit according to one embodiment of the present invention is a kit for detecting or quantifying an analyte contained in a sample using, for example, a test strip 100 based on the analyte measurement method of the present invention.

[0062] The analyte measurement kit of the present invention includes a test strip 100. The analyte measurement kit of the present invention may further include other components as necessary. For example, the analyte measurement kit may include a detection reagent containing a labeled antibody in which an antibody that specifically binds to the analyte is labeled with a labeling substance. The analyte measurement kit may also include a measuring device that measures the color intensity in the determination unit 20. The analyte measurement kit may also include reference standard data such as a calibration curve, table, or formula showing the relationship between color intensity and analyte concentration. The analyte measurement kit may also include a developing solution.

[0063] When using the analyte measurement kit of the present invention, the analyte in the sample may be brought into contact with the labeled antibody in the detection reagent to perform step (I), and then the sample may be applied to the test strip 100, and steps (II) to (V) may be performed sequentially. Alternatively, the sample may be applied to the sample application section 40 or reaction section 50 formed upstream of the determination section 20 of the test strip 100, and steps (I) to (V) may be performed sequentially in the test strip 100. [Example]

[0064] 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.

[0065] <Absorbance measurement of platinum-resin composite particles> The absorbance of the platinum-resin composite particles was measured by placing a platinum-resin composite particle dispersion (dispersion medium: water) prepared at 0.01 wt% (weight %) in a quartz glass cell (light path length 10 mm) and using a spectrophotometer (Shimadzu Corporation, UV3600) to measure the absorbance at 400 nm. <Absorbance measurement of gold-resin composite particles> The absorbance of the gold-resin composite particles was measured by placing a gold-resin composite particle dispersion (dispersion medium: water) prepared at 0.01 wt% (weight %) in a quartz glass cell (light path length 10 mm) and using a spectrophotometer (Shimadzu Corporation, UV3600) to measure the absorbance at 570 nm.

[0066] <Measurement of solid concentration and metal loading amount> 1 g of the dispersion of metal-resin composite particles before concentration adjustment was placed in a porcelain crucible and dried for 3 hours at 70° C. The weights before and after drying were measured, 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 3 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 (wt%) = [Weight after heat treatment (g) / Weight before heat treatment (g)] x 100

[0067] <Measurement of 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.

[0068] <Measurement of the average particle size of resin particles and metal-resin composite particles> Measurement was performed using a centrifugal sedimentation particle size distribution analyzer (LUMiSizer610 manufactured by LUM GmbH) with the particles dispersed in water or a solution.

[0069] <Calculation of particle capture yield> Using a mock test strip (mock test strip 100A shown in Figure 2) with four control lines CL1, CL2, CL3, and CL4 as described in the test example, 50 μl of developing solution 1 (antigen-free) was dropped onto the sample pad of the mock test strip. After 30 minutes, measurements were taken using an immunochromatography reader (Hamamatsu Photonics C10066-10), and the sum of the color intensities of the four control lines CL1, CL2, CL3, and CL4 was taken as the initial total amount Q of labeled antibody. When the color intensity of control line CL1 is CL1, the color intensity of control line CL2 is CL2, the color intensity of control line CL3 is CL3, and the color intensity of control line CL4 is CL4, the particle capture yield was calculated using the following formula: Particle capture yield up to CL1 (%) = [CL1 / Q] × 100 Particle capture yield up to CL2 (%) = (CL1 + CL2) / Q × 100 Particle capture yield up to CL3 (%) = (CL1 + CL2 + CL3) / Q × 100 Particle capture yield up to CL4 (%) = (CL1 + CL2 + CL3 + CL4) / Q × 100

[0070] [Production Example 1] <Synthesis of resin particles> A 500 mL round vessel was equipped with an incomplete semicircular stirring blade, with its longitudinal axis perpendicular to the vessel's center. 300 g of purified water, an 80 wt% aqueous solution of trioctylmethylammonium chloride (1.55 g, 3.07 mmol), and a 50 wt% aqueous solution of polyethylene glycol methyl ether methacrylate (10.00 g, 2.40 mmol) were mixed in the vessel. Next, 2-vinylpyridine (48.00 g, 457 mmol) and divinylbenzene (2.00 g, 15.4 mmol) were added, and the mixture was stirred under a nitrogen stream at 30°C for 50 minutes, followed by 30 minutes at 60°C. After stirring, 2,2-azobis(2-methylpropionamidine) dihydrochloride (0.250 g, 0.922 mmol) dissolved in 18.00 g of pure water was added dropwise and stirred at 60°C for 3 hours to obtain resin particles A-1. After precipitation by centrifugation and removal of the supernatant, the particles were redispersed in pure water and filtered to remove impurities. The concentration was then adjusted to obtain a 10 wt% resin particle dispersion B-1. The average particle diameter of the resin particles A-1 in the resin particle dispersion B-1 was 395 nm.

[0071] [Production Example 2] <Synthesis of resin particles> A 500 mL round vessel was equipped with an incomplete semicircular stirring blade, with its longitudinal axis perpendicular to the vessel's center. 300 g of purified water, an 80 wt% aqueous solution of trioctylmethylammonium chloride (1.76 g, 3.48 mmol), and a 50 wt% aqueous solution of polyethylene glycol methyl ether methacrylate (10.00 g, 2.40 mmol) were mixed in the vessel. 2-Vinylpyridine (48.00 g, 457 mmol) and divinylbenzene (2.00 g, 15.4 mmol) were then added. 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 (0.250 g, 0.922 mmol) dissolved in 18.00 g of pure water was added dropwise and stirred at 60°C for 3 hours to obtain resin particles A-2. After precipitation by centrifugation and removal of the supernatant, the particles were redispersed in pure water and filtered to remove impurities. The concentration was then adjusted to obtain 10 wt% resin particle dispersion B-2. The average particle diameter of resin particles A-2 in resin particle dispersion B-2 was 328 nm.

[0072] [Preparation Example 3] <Synthesis of platinum-resin composite particles> 83.5 g of pure water was added to B-1 (30.00 g) obtained in Preparation Example 1, followed by addition of a 7 wt % aqueous solution of chloroplatinic acid (31.5 g) and stirring at 30°C for 3 hours. This mixture was centrifuged and the supernatant was removed to remove excess chloroplatinic acid. The concentration was then adjusted to obtain a 5 wt % platinum ion-adsorbing resin particle dispersion C-1.

[0073] Next, 70.45 g of C-1 diluted with 34.2 g of pure water was added to 4725 g of pure water with stirring at 3°C. Furthermore, 138 g of 132 mM dimethylamine borane solution was added dropwise over 20 minutes with stirring at 3°C. The mixture was then stirred at 3°C ​​for 60 minutes and then at room temperature for 2 hours to obtain platinum-resin composite particles D-1 with an average particle size of 438 nm. D-1 was concentrated and purified by dialysis, and the concentration was adjusted to obtain a 1 wt% platinum-resin composite particle dispersion E-1. The absorbance of platinum-resin composite particles F-1 in E-1 was 1.77. The average particle size of the platinum particles in F-1 was 3 nm, the standard deviation of the platinum particle size was 0.9 nm, and the platinum loading was 39.9 wt%. In this platinum-resin composite particle F-1, the platinum particles include encapsulated platinum particles that are completely encapsulated in the resin particles, and partially exposed platinum particles that have portions embedded within the resin particles and portions exposed outside the resin particles, and at least some of the platinum particles are distributed three-dimensionally in the surface layer portion of the resin particles.

[0074] [Preparation Example 4] <Synthesis of platinum-resin composite particles> A 5 wt% platinum ion-adsorbing resin particle dispersion C-2 was obtained in the same manner as in Preparation Example 3, except that B-2 obtained in Preparation Example 2 was used instead of B-1. Subsequently, platinum-resin composite particles D-2 with an average particle diameter of 368 nm were obtained in the same manner as in Preparation Example 3. D-2 was concentrated and then purified by dialysis, and the concentration was adjusted to obtain a 1 wt% platinum-resin composite particle dispersion E-2. The absorbance of platinum-resin composite particles F-2 in E-2 was 1.64. The average particle diameter of the platinum particles in F-2 was 3 nm, the standard deviation of the platinum particle diameters was 0.9 nm, and the platinum loading was 37.5 wt%. In this platinum-resin composite particle F-2, the platinum particles include encapsulated platinum particles that are completely encapsulated in the resin particles and partially exposed platinum particles that have portions embedded within the resin particles and portions exposed outside the resin particles, and at least some of the platinum particles are distributed three-dimensionally in the surface layer portion of the resin particles.

[0075] [Preparation Example 5] <Synthesis of gold-resin composite particles> 75.00 g of pure water was added to 30.00 g of B-2 obtained in Preparation Example 2, followed by 42.9 g of a 7 wt% aqueous solution of chloroauric acid. The mixture was then centrifuged and the supernatant was removed to remove excess chloroauric acid. The concentration was then adjusted to obtain a 5 wt% dispersion of gold ion-adsorbing resin particles, C-3.

[0076] Next, 65.01 g of C-3 diluted with 30.0 g of pure water was added to 4725 g of pure water at 3°C ​​while stirring. Furthermore, 15.5 g of 528 mM dimethylamine borane solution was added dropwise over 53 seconds while stirring at 3°C. The mixture was then stirred at 3°C ​​for 60 minutes and then at room temperature for 2 hours to obtain gold-resin composite particles D-3 with an average particle size of 342 nm. D-3 was concentrated and purified by dialysis, and the concentration was adjusted to obtain a 1 wt% gold-resin composite particle dispersion E-3. The absorbance of gold-resin composite particles F-3 in E-3 was 1.36. The average particle size of the gold particles in F-3 was 19.2 nm, the standard deviation of the gold particle size was 31.1 nm, and the gold loading was 48.3 wt%. In this gold-resin composite particle F-3, the gold particles included encapsulated gold particles that were completely encapsulated in the resin particles and partially exposed gold particles that had portions embedded within the resin particles and portions exposed outside the resin particles, and at least some of the gold particles were distributed three-dimensionally on the surface layer of the resin particles.

[0077] [Test Example 1] (Antibody binding process) After mixing 25 μg of anti-CRP antibody with 0.45 mL of 50 mM HEPES buffer (pH 7), 0.05 mL of 1 wt% platinum-resin composite particle dispersion E-1 was added, and the mixture was stirred end-over-end at room temperature for 1 hour to obtain labeled antibody dispersion G-1 containing anti-CRP antibody labeled with platinum-resin composite particle F-1.

[0078] (Blocking process) Next, the labeled antibody dispersion G-1 was centrifuged at 3000 rpm for 5 minutes, and the supernatant was removed. 0.5 mL of HEPES buffer (pH 7) containing 1 wt% sodium caseinate was added to the sediment, which was then ultrasonically dispersed. The mixture was then stirred end-over-end at room temperature for 1 hour to obtain labeled antibody dispersion H-1.

[0079] (Cleaning process) Next, the labeled antibody dispersion H-1 was centrifuged at 3000 rpm for 5 minutes, and the supernatant was removed. Then, 0.5 mL of a 5 mM Tris aqueous solution (pH 8.5) containing less than 0.1 wt% surfactant was added to the sediment, followed by ultrasonic dispersion. This procedure was repeated three times to obtain labeled antibody dispersion I-1.

[0080] (Preparation of conjugate pad) Labeled antibody dispersion I-1 was centrifuged at 3000 rpm for 5 minutes, and the supernatant was removed. A 5 mM Tris aqueous solution (pH 8.5) containing 5 wt% sucrose and 2.5 wt% BSA was then added to the sediment and ultrasonically dispersed to obtain labeled antibody dispersion J-1. A glass fiber nonwoven fabric was uniformly impregnated with labeled antibody dispersion J-1 and then dried at 50°C for 1 hour to produce conjugate pad K-1. The concentration of labeled antibody dispersion J-1 and the amount applied to the glass fiber nonwoven fabric were adjusted so that the content of platinum-resin composite particles F-1 in conjugate pad K-1 was 3 μg per test evaluated by the immunochromatography method described below.

[0081] (Preparation of immunochromatographic test strips) To investigate the relationship between the capture yield of labeled antibodies and the number of control lines, a simulated test strip 100A with the structure shown in Figure 2 was prepared. First, a 25 mm-wide nitrocellulose membrane (membrane 10) was coated with 0.1 mg / ml anti-mouse IgG antibody at a coating volume of 1 μL / cm to create four control lines CL1, CL2, CL3, and CL4, forming the control zone 30. The control lines CL1, CL2, CL3, and CL4 were spaced equally at 3 mm intervals. No test line was provided as the determination zone 20. This nitrocellulose membrane was dried at 50°C for 1 hour, and then laminated with a laminate film (support 70), a conjugate pad K-1 (reaction zone 50), a sample pad (glass fiber nonwoven fabric) (sample addition zone 40), and an absorbent pad (cotton nonwoven fabric) (liquid-absorbing zone 60) as shown in the cross-sectional view of Figure 2. Finally, the membrane was cut to a width of 3.5 mm to form the simulated test strip 100A.

[0082] (Preparation of developing solution) An aqueous solution (pH 7.5) containing 50 mM Tris, 150 mM NaCl, 1.0 wt % BSA, and 1.0 wt % surfactant Triton X-100 was prepared and used as developing solution 1.

[0083] (Evaluation by immunochromatography) 50 μl of developing solution 1 (antigen-free) was dropped onto the sample pad of the mock test strip 100A. After 30 minutes, the color intensity of the four control lines CL1, CL2, CL3, and CL4 was measured using an immunochromatograph (Hamamatsu Photonics C10066-10). The evaluation results are shown in Table 1.

[0084] [Test Example 2] Except for the fact that the concentration of anti-mouse IgG antibody applied to the four control lines CL1, CL2, CL3, and CL4 was 0.25 mg / ml, the antibody binding process, blocking process, washing treatment, conjugate pad preparation, mock test strip preparation, and immunochromatographic evaluation were carried out in the same manner as in Test Example 1. The results are shown in Table 1.

[0085] [Test Example 3] Except for the fact that the concentration of anti-mouse IgG antibody applied to the four control lines CL1, CL2, CL3, and CL4 was 0.5 mg / ml, the antibody binding process, blocking process, washing treatment, conjugate pad preparation, mock test strip preparation, and immunochromatographic evaluation were carried out in the same manner as in Test Example 1. The results are shown in Table 1.

[0086] [Test Example 4] Except for the fact that the concentration of anti-mouse IgG antibody applied to the four control lines CL1, CL2, CL3, and CL4 was 1 mg / ml, the antibody binding process, blocking process, washing treatment, conjugate pad preparation, mock test strip preparation, and immunochromatographic evaluation were carried out in the same manner as in Test Example 1. The results are shown in Table 1.

[0087] [Test Example 5] Except for the fact that the concentration of anti-mouse IgG antibody applied to the four control lines CL1, CL2, CL3, and CL4 was 1.5 mg / ml, the antibody binding process, blocking process, washing treatment, conjugate pad preparation, mock test strip preparation, and immunochromatographic evaluation were carried out in the same manner as in Test Example 1. The results are shown in Table 1.

[0088] [Test Example 6] Except for using 1 wt% platinum-resin composite particle dispersion E-2 instead of 1 wt% platinum-resin composite particle dispersion E-1 and changing the concentration of anti-mouse IgG antibody applied to the four control lines CL1, CL2, CL3, and CL4 to 1 mg / ml, the antibody binding step, blocking step, washing treatment, conjugate pad preparation, mock test strip preparation, and immunochromatographic evaluation were performed in the same manner as in Test Example 1. The results are shown in Table 1.

[0089] [Test Example 7] Except for using 1 wt% gold-resin composite particle dispersion E-3 instead of 1 wt% platinum-resin composite particle dispersion E-1 and changing the concentration of anti-mouse IgG antibody applied to the four control lines CL1, CL2, CL3, and CL4 to 1 mg / ml, the antibody binding step, blocking step, washing treatment, conjugate pad preparation, mock test strip preparation, and immunochromatographic evaluation were performed in the same manner as in Test Example 1. The results are shown in Table 1.

[0090] [Test Example 8] The antibody binding process, blocking process, washing process, conjugate pad preparation, mock test strip preparation, and immunochromatographic evaluation were performed as in Test Example 1, except that gold colloid (Tanaka Kikinzoku Kogyo, Au Colloid Solution, product name: Au Colloid Solution-SC, particle size 30 nm) was used as the labeled particles, 5 mM Tris aqueous solution (pH 9.5) was used as the binding buffer, and 5 mM Tris aqueous solution (pH 9.5) containing 1 wt% BSA was used as the blocking buffer, the concentration of anti-mouse IgG antibody applied to the four control lines CL1, CL2, CL3, and CL4 was 1 mg / ml, and the gold colloid content in the conjugate pad was 1 μg per immunochromatographic evaluation test. The results are shown in Table 1.

[0091] [Table 1]

[0092] As shown in Table 1, by providing four control lines, it was possible to increase the particle capture yield to 90% or more, regardless of the type or concentration of labeled antibody. The number of control lines required to achieve a particle capture yield of 90% or more varied depending on the concentration of antibody applied to the control lines: four in Test Example 1 (antibody concentration 0.1 mg / ml), three in Test Example 2 (antibody concentration 0.25 mg / ml), two in Test Example 3 (antibody concentration 0.5 mg / ml), and two in Test Example 4 (antibody concentration 1 mg / ml). On the other hand, in Test Example 5 (antibody concentration 1.5 mg / ml), the color intensity of CL1 measured with the immunochromatographic reader exceeded the upper limit of 800 mABS that could be quantitatively measured according to the specifications of the measuring device, making quantitative measurement difficult.

[0093] [Example 1] 1 wt% platinum-resin composite particle dispersion E-1 was subjected to the antibody binding process, blocking process, and washing process in the same manner as in Test Example 1, and conjugate pad K-1 was prepared in the same manner as in Test Example 1.

[0094] (Preparation of immunochromatographic test strips) A test strip was prepared with the same structure as shown in Figure 1, except that the number of control lines was three. First, a 25 mm wide nitrocellulose membrane (membrane 10) was coated with an anti-CRP antibody at a concentration of 0.5 mg / ml to form a test line as the test zone 20. Additionally, a 0.25 mg / ml anti-mouse IgG antibody was applied in a linear fashion at a rate of 1 μL / cm downstream of the test line to form three control lines CL1, CL2, and CL3, which served as the control zone 30. The control lines CL1, CL2, and CL3 were spaced equally apart at 3 mm. After drying the nitrocellulose membrane at 50°C for 1 hour, a laminate film (support 70), a conjugate pad K-1 (reaction zone 50), a sample pad (glass fiber nonwoven fabric) as the sample addition zone 40, and an absorbent pad (cotton nonwoven fabric) as the liquid-absorbing zone 60 were laminated on top of the membrane. Finally, the membrane was cut to a width of 3.5 mm to form a test strip.

[0095] (Evaluation by immunochromatography) A positive control sample solution (antigen concentration 19.5 ng / ml) was prepared by diluting the CRP antigen using the same developer solution 1 as in Test Example 1. 50 μl of the sample solution was dropped onto the sample pad of the test strip. After 30 minutes, the color intensity of the test line and control lines CL1, CL2, and CL3 was measured using an immunochromatograph (Hamamatsu Photonics C10066-10). Five measurements were performed on the same sample solution, and after correction based on formula (2), the CV value (coefficient of variation) representing variability was calculated. The results are shown in Table 2.

[0096] [Example 2] Except for the use of two control lines and the use of 0.5 mg / ml of anti-mouse IgG antibody applied to each of the control lines CL1 and CL2, the antibody binding step, blocking step, washing treatment, conjugate pad preparation, test strip preparation, and immunochromatographic evaluation were carried out in the same manner as in Example 1. The results are shown in Table 2.

[0097] [Example 3] Except for using two control lines and applying anti-mouse IgG antibodies to each of the control lines CL1 and CL2 at a concentration of 1 mg / ml, the antibody binding step, blocking step, washing treatment, conjugate pad preparation, test strip preparation, and immunochromatographic evaluation were carried out in the same manner as in Example 1. The results are shown in Table 2.

[0098] [Example 4] The antibody binding step, blocking step, washing treatment, conjugate pad preparation, test strip preparation, and immunochromatographic evaluation were carried out in the same manner as in Example 1, except that 1 wt % platinum-resin composite particle dispersion E-2 was used instead of 1 wt % platinum-resin composite particle dispersion E-1, two control lines were used, and the concentration of anti-mouse IgG antibody applied to each control line CL1 and CL2 was 1 mg / ml. The results are shown in Table 2.

[0099] [Example 5] The antibody binding step, blocking step, washing treatment, conjugate pad preparation, test strip preparation, and immunochromatographic evaluation were carried out in the same manner as in Example 1, except that 1 wt % gold-resin composite particle dispersion E-3 was used instead of 1 wt % platinum-resin composite particle dispersion E-1, the number of control lines was two, and the concentration of anti-mouse IgG antibody applied to each control line CL1 and CL2 was 1 mg / ml. The results are shown in Table 2.

[0100] [Example 6] The antibody binding step, blocking step, washing process, conjugate pad preparation, test strip preparation, and immunochromatographic evaluation were carried out as in Example 1, except that gold colloid (Tanaka Kikinzoku Kogyo, Au Colloid Solution, product name: Au Colloid Solution-SC, particle size 30 nm) was used as the labeled particles, 5 mM Tris aqueous solution (pH 9.5) was used as the binding buffer, and 5 mM Tris aqueous solution (pH 9.5) containing 1 wt% BSA was used as the blocking buffer. Two control lines were used, and the anti-mouse IgG antibody concentration applied to each control line CL1 and CL2 was 1 mg / ml. The gold colloid content in the conjugate pad was 1 μg per immunochromatographic evaluation test. The results are shown in Table 2.

[0101] [Table 2]

[0102] [Comparative Example 1] Except for using one control line, the antibody binding step, blocking step, washing treatment, conjugate pad preparation, test strip preparation, and evaluation by immunochromatography were performed in the same manner as in Example 1. The results are shown in Table 3.

[0103] Comparative Example 2 Except for using one control line, the antibody binding step, blocking step, washing treatment, conjugate pad preparation, test strip preparation, and evaluation by immunochromatography were performed in the same manner as in Example 2. The results are shown in Table 3.

[0104] Comparative Example 3 Except for using one control line, the antibody binding step, blocking step, washing treatment, conjugate pad preparation, test strip preparation, and evaluation by immunochromatography were performed in the same manner as in Example 3. The results are shown in Table 3.

[0105] Comparative Example 4 Except for using one control line, the antibody binding step, blocking step, washing treatment, conjugate pad preparation, test strip preparation, and evaluation by immunochromatography were performed in the same manner as in Example 4. The results are shown in Table 3.

[0106] Comparative Example 5 Except for using one control line, the antibody binding step, blocking step, washing treatment, conjugate pad preparation, test strip preparation, and evaluation by immunochromatography were performed in the same manner as in Example 5. The results are shown in Table 3.

[0107] Comparative Example 6 Except for using one control line, the antibody binding step, blocking step, washing treatment, conjugate pad preparation, test strip preparation, and evaluation by immunochromatography were performed in the same manner as in Example 6. The results are shown in Table 3.

[0108] [Table 3]

[0109] A comparison of Tables 2 and 3 shows that in Examples 1 to 6, by providing multiple control lines and performing correction based on formula (2), the CV values, which represent the variability of the measurement data, are significantly smaller than in Comparative Examples 1 to 6, confirming that highly accurate quantification of the analyte is possible.

[0110] 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. [Explanation of symbols]

[0111] 10...membrane, 20...determination section, 30...control section, 40...sample addition section, 50...reaction section, 60...liquid absorption section, 100...test strip, 100A...dummy test strip, CL1, CL2, CL3, CL4...control lines

Claims

1. A lateral flow chromatographic test strip for detecting or quantifying an analyte contained in a sample, comprising: a determination unit having a first capture ligand immobilized thereon that specifically binds to the analyte; a control section, located downstream of the determination section in the direction of development of the sample, in which a second capture ligand is immobilized that specifically binds to a labeled antibody obtained by labeling an antibody that specifically binds to the analyte with a labeling substance; Equipped with A lateral flow chromatography test strip, characterized in that the control section has a plurality of control lines spaced apart in the direction in which the sample develops.

2. 2. The lateral flow chromatography test strip according to claim 1, wherein the number of the control lines is in the range of 2 to 5.

3. 2. The lateral flow chromatography test strip according to claim 1, wherein the amount of the second capture ligand immobilized on the plurality of control lines is equal.

4. 2. The lateral flow chromatography test strip according to claim 1, wherein the amount of the second capture ligand immobilized on the plurality of control lines is different.

5. 2. The lateral flow chromatography test strip according to claim 1, wherein when there are three or more control lines, the control lines are arranged at equal intervals in the direction in which the sample develops.

6. 2. The lateral flow chromatography test strip according to claim 1, wherein when there are three or more control lines, the control lines are arranged at different intervals in the direction in which the sample develops.

7. a reaction zone containing the labeled antibody is provided upstream of the determination zone; The amount Q2 that can be captured by the second capture ligand relative to the initial total amount Q1 of the labeled antibody in the reaction zone is 0.9×Q1≦Q2≦1.0×Q1 2. The lateral flow chromatography test strip according to claim 1, wherein the following relationship is satisfied:

8. The lateral flow chromatography test strip according to claim 1, wherein the labeling substance is a labeling substance comprising a metal-resin complex having a resin particle and a plurality of metal particles immobilized on the resin particle.

9. A method for measuring an analyte by detecting or quantifying an analyte contained in a sample using a lateral flow chromatography test strip, comprising: The lateral flow chromatography test strip according to any one of claims 1 to 8 is used, and the following steps (I) to (V) are carried out: Step (I): contacting the sample with a labeled antibody obtained by labeling an antibody that specifically binds to the analyte with a labeling substance; Step (II): contacting the conjugate of the analyte and the labeled antibody with the first capture ligand in the determination section; Step (III): contacting the labeled antibody and / or the conjugate with the second capture ligand in the control section; Step (IV): measuring the color intensity caused by the labeling substance in the judgment section and the control section, respectively; Step (V): quantitating the amount of analyte contained in the sample based on the measured color intensity; A method for measuring an analyte, comprising:

10. 10. The method for measuring an analyte according to claim 9, wherein in step (V), the color intensity in the judgment zone is corrected by dividing it by the sum of the color intensities in the judgment zone and the control zone, and then the amount of analyte contained in the sample is estimated.

11. The method for measuring an analyte according to claim 9, wherein the sum of the amount of labeled antibody in the conjugate captured by the first capture ligand in step (II) and the amount of labeled antibody captured by the second capture ligand in step (III) is 90% or more of the initial total amount of labeled antibody used in step (I).

12. An analyte measurement kit for detecting or quantifying an analyte contained in a sample, comprising: An analyte measurement kit comprising the lateral flow chromatography test strip according to any one of claims 1 to 8.

13. The analyte measurement kit according to claim 12, further comprising reference standard data showing the relationship between the color intensity or a corrected value thereof in the determination zone and the concentration of the analyte.

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