Immunological test method

Abstract

The present invention addresses the problem of providing an immunological testing method which is highly sensitive and in which false positives are unlikely to occur. This immunological testing method comprises: a concentration step in which a liquid that could contain an antigen is mixed with a highly water-absorbent polymer to concentrate the liquid that could contain an antigen and to obtain an antigen-concentrated liquid; and a detection step in which an antigen in the antigen-concentrated liquid is detected by using an antigen-antibody reaction. The content, in the antigen-concentrated liquid, of a polymer having a weight-average molecular weight of less than 1,000,000 is more than 0 mg / mL and less than 35 mg / mL.

Description

Immunoassay method

[0001] The present invention relates to an immunoassay method.

[0002] Immunoassay methods (especially immunochromatography) have been frequently used in recent years due to their simple operation and ability to perform measurements in a short time. For example, when detecting an antigen such as an influenza virus antigen using immunochromatography, the following procedure is performed. First, a label modified with an antibody (labeled antibody) is prepared and mixed with a liquid that may contain the antigen (hereinafter also referred to as a "sample liquid"). The labeled antibody binds to the antigen to form a complex. In this state, the complex is developed on an insoluble carrier having a detection line coated with an antibody that specifically reacts with the antigen, where it reacts with the antibody and is captured on the detection line (test line), and detection can be confirmed visually or otherwise.

[0003] Recently, there has been a demand for immunoassay methods that can be applied to sample liquids with extremely low antigen concentrations. To address this, a method has been proposed in which the sample liquid is concentrated using a highly water-absorbent polymer before detecting the antigen (Patent Document 1).

[0004] Patent No. 7192146

[0005] Under these circumstances, the present inventors have investigated the method described in Patent Document 1 and found that it may not always provide sufficient sensitivity, and that false positives may occur.

[0006] In view of the above circumstances, an object of the present invention is to provide an immunoassay method that is highly sensitive and less likely to produce false positives.

[0007] The inventors' investigations have revealed that uncrosslinked polymers present in superabsorbent polymers are mixed into the antigen concentrate, affecting sensitivity and false positives, and that by setting the content of the uncrosslinked polymer in the antigen concentrate within a predetermined range, sensitivity is improved and false positives are less likely to occur. The present invention is based on the above findings and aims to solve the above problems by the following configuration.

[0008] (1) An immunoassay method comprising: a concentrating step of concentrating a liquid that may contain an antigen by mixing the liquid with a superabsorbent polymer to obtain an antigen concentrated solution; and a detecting step of detecting the antigen in the antigen concentrated solution using an antigen-antibody reaction, wherein the content of polymers having a weight-average molecular weight of less than 1,000,000 in the antigen concentrated solution is more than 0 mg / mL and less than 35 mg / mL. (2) The immunoassay method according to (1) above, wherein the superabsorbent polymer is a polyacrylic acid-based or polyacrylamide-based polymer. (3) The immunoassay method according to (1) or (2) above, wherein the polymer is at least one polymer or copolymer selected from the group consisting of acrylic acid, sodium acrylate, and acrylic acid amide.

[0009] As will be shown below, the present invention can provide an immunoassay method that is highly sensitive and less likely to produce false positives.

[0010] FIG. 1 is a diagram conceptually illustrating an example of a concentration device of the present invention. FIG. 2 is a diagram for explaining the operation of the concentration device shown in FIG. 1. FIG. 3 is a diagram for explaining the operation of the concentration device shown in FIG. 1. FIG. 4 is a diagram for explaining the operation of the concentration device shown in FIG. 1. FIG. 5 is a perspective view schematically illustrating an example of a concentration device of the present invention. FIG. 6 is an exploded perspective view of the concentration device shown in FIG. 7. FIG. 7 is a perspective view schematically illustrating an example of a cap included in the concentration device of the present invention. FIG. 8 is a perspective view schematically illustrating another example of a container body included in the concentration device of the present invention. FIG. 9 is a perspective view schematically illustrating another example of a container body included in the concentration device of the present invention. FIG. 10 is a diagram for explaining the relationship between the discharge direction of a liquid sample and a soft wall surface. FIG. 11 is a diagram for explaining the possible change in volume of a container. FIG. 12 is a schematic view of one embodiment of an insoluble carrier used in the method of the present invention. １ 1H-NMR spectrum. GPC chromatogram of the supernatant of the concentrated solution. Immunochromatography BG plot against polymer concentration.

[0011] The immunoassay method of the present invention will be described below. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits. In this specification, each component may be used singly or in combination of two or more. When two or more components are used in combination, the content of the components refers to the total content unless otherwise specified. High sensitivity is also referred to as "excellent sensitivity." High sensitivity and low false positives are also collectively referred to as "excellent effects of the present invention."

[0012] [1] Immunoassay method The immunoassay method of the present invention (hereinafter also referred to as "the method of the present invention") comprises: a concentration step of concentrating a liquid that may contain an antigen by mixing the liquid with a superabsorbent polymer to obtain an antigen concentrate; and a detection step of detecting the antigen in the antigen concentrate using an antigen-antibody reaction, wherein the content of polymers having a weight-average molecular weight of less than 1,000,000 in the antigen concentrate is more than 0 mg / mL and less than 35 mg / mL.

[0013] Each step of the method of the present invention will be described below.

[0014] [Concentration Step] The concentration step is a step of concentrating a liquid that may contain an antigen (sample liquid) by mixing the liquid with a highly water-absorbent polymer, thereby obtaining an antigen concentrate.

[0015] [Sample Liquid] The sample liquid used in the concentration step is not particularly limited as long as it is a liquid that can contain an antigen. Examples of such liquids include biological samples, particularly animal (e.g., human) body fluids (e.g., blood, serum, plasma, cerebrospinal fluid, tears, sweat, urine, pus, nasal discharge, or sputum), gargle, etc.

[0016] The sample liquid is preferably urine, because the effects of the present invention are more excellent. When the sample liquid is urine, the concentration of urea in the antigen concentrate obtained in the concentration step is preferably 5 times or less the concentration of urea in the sample liquid, because the effects of the present invention are more excellent.

[0017] <Antigen> Examples of antigens include fungi, bacteria (e.g., Mycobacterium tuberculosis, lipoarabinomannan (LAM) contained in Mycobacterium tuberculosis), bacteria, viruses (e.g., influenza virus), and their nucleoproteins. LAM is a major antigen in tuberculosis and is a glycolipid that is a major component of cell membranes and cell walls. For reasons of superior effect of the present invention, the antigen is more preferably a virus (particularly influenza virus) or LAM, and even more preferably LAM.

[0018] <Pretreatment of Sample Solution> The sample solution can be used as is, or in the form of an extract obtained by extracting the antigen with an appropriate extraction solvent, or in the form of a diluted solution obtained by diluting the extract with an appropriate diluent, or in the form of an extract concentrated by an appropriate method. The extraction solvent can be a solvent used in conventional immunological analysis methods (e.g., water, physiological saline, buffer solution, etc.), or a water-miscible organic solvent that can be diluted with such a solvent to directly carry out an antigen-antibody reaction.

[0019] [Superabsorbent polymer] The superabsorbent polymer (hereinafter also referred to as "SAP") used in the concentration step is not particularly limited. Note that SAP is usually a crosslinked polymer, and its molecular weight cannot be measured.

[0020] <Preferred embodiment> The polymer constituting the superabsorbent polymer is preferably a polyacrylic acid-based, polyacrylamide-based, cellulose-based, or polyethylene oxide-based polymer, and more preferably a polyacrylic acid-based or polyacrylamide-based polymer, because this provides a more excellent effect of the present invention.

[0021] <Swelling ratio> For reasons of superior effects of the present invention, the swelling ratio of the superabsorbent polymer is preferably more than 0.2 g / g and less than 800 g / g, more preferably 1.0 g / g or more and 600 g / g or less, even more preferably 10 g / g or more and 400 g / g or less, and particularly preferably 20 g / g or more and 200 g / g or less. Here, the swelling ratio is a value defined as "the mass (g) of water held by 1 g of superabsorbent polymer."

[0022] (Method for measuring swelling ratio) The mass of a superabsorbent polymer stored at 25°C and 5% RH (relative humidity) for 10 days is measured, and immediately thereafter, the polymer is immersed in a large amount of distilled water. After 120 minutes, the superabsorbent polymer is removed, the water on the surface is removed, the mass is measured again, and the swelling ratio is calculated using the following formula: {(mass after water absorption (g) - initial mass before water absorption (g)) / initial mass before water absorption (g)}

[0023] The method for adjusting the swelling ratio to fall within the above-mentioned specific range is not particularly limited, but examples thereof include changing the type of polymer, changing the degree of crosslinking, and changing the particle size.

[0024] <Particle size> The superabsorbent polymer is preferably in a particulate form, and in this case, the particle size is preferably 5 mm or less, more preferably 3 mm or less, and even more preferably 1.6 mm or less, because the effects of the present invention are more excellent. The lower limit of the particle size of the superabsorbent polymer is preferably 0.01 mm or more, more preferably 0.1 mm or more, and even more preferably 0.3 mm or more, because the effects of the present invention are more excellent. The particle size is generally not uniform but has a distribution. The particle size can be determined by measuring the diameters of 50 particulate polymers using an optical microscope and calculating the arithmetic average value.

[0025] <Amount Used> The amount of the superabsorbent polymer used is not particularly limited, but in order to obtain a more excellent effect of the present invention, it is preferably 0.001 to 10 g, and more preferably 0.01 to 1 g, per mL of sample liquid.

[0026] [Procedure for Concentration Step] The procedure for the concentration step is not particularly limited, and examples thereof include a method of mixing the sample liquid with a superabsorbent polymer and recovering the sample liquid (antigen concentrate) that has not been absorbed by the superabsorbent polymer. The method for mixing the sample liquid with the superabsorbent polymer is also not particularly limited, and examples thereof include a method of blending the superabsorbent polymer with the sample liquid, stirring, and allowing to stand. If it is difficult to remove the antigen concentrate, it is also preferable to add a recovery liquid (sample liquid, water, etc.) in an amount smaller than the sample liquid before recovery. For the concentration step, it is preferable to use a concentration tool described below, because this provides better effects of the present invention.

[0027] [Detection Step] The detection step is a step of detecting the antigen in the antigen concentrate obtained in the concentration step described above using an antigen-antibody reaction.

[0028] The detection step is not particularly limited as long as it uses an antigen-antibody reaction, and examples thereof include enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), Western blotting, immunochromatography, etc. Among these, immunochromatography is preferred because it provides superior effects of the present invention. That is, the method of the present invention is preferably immunochromatography.

[0029] [Concentrated Antigen Solution] In the detection step, the concentrated antigen solution obtained in the concentration step described above is used.

[0030] <Polymer> The antigen concentrate contains a polymer with a weight-average molecular weight (Mw) of less than 1,000,000. The Mw is a value measured by gel permeation chromatography (GPC). The polymer may be an uncrosslinked polymer contained in the SAP used in the concentration step that has been mixed into the antigen concentrate, or may be a polymer added separately. The polymer concentration in the antigen concentrate can be calculated by measuring the absorbance with an absorptiometer using a standard polymer solution having a molecular weight equivalent to that obtained by GPC and creating a calibration curve.

[0031] (Preferred embodiment) For the reason that the effects of the present invention are more excellent, the polymer is preferably at least one polymer or copolymer (hereinafter also referred to as "specific polymer") selected from the group consisting of acrylic acid, sodium acrylate, and acrylic acid amide. Specific examples of the specific polymer include polyacrylic acid, polyacrylic acid amide, and a copolymer of acrylamide and acrylic acid.

[0032] <Polymer concentration> The content of polymers with a weight-average molecular weight of less than 1,000,000 in the antigen concentrate is more than 0 mg / mL and less than 35 mg / mL. Hereinafter, the content [mg / mL] of polymers with a weight-average molecular weight (Mw) of less than 1,000,000 in the antigen concentrate will also be referred to as the "polymer concentration."

[0033] The polymer concentration in the concentrated antigen solution is preferably 1 mg / mL or more, and more preferably 5 mg / mL or more, for reasons of better effects of the present invention.

[0034] There are no particular limitations on the method for adjusting the polymer concentration in the antigen concentrate to the above-mentioned range. However, when the polymer is an uncrosslinked polymer contained in the SAP used in the concentration step that has become mixed into the antigen concentrate, examples of methods that can be used include adjusting the amounts of the sample solution, SAP, and recovery solution used in the concentration step.

[0035] [Preferred Embodiment] A preferred embodiment in which the detection step is immunochromatography is described below. For reasons that provide better effects of the present invention, the detection step preferably includes: a development step in which a gold particle complex, which is a complex between the antigen in the antigen concentrate and modified gold particles (gold particles modified with a first binding substance capable of binding to the antigen), is formed, and the gold particle complex is developed on an insoluble carrier having a reaction site at which a second binding substance capable of binding to the antigen is immobilized; a capture step in which the gold particle complex is captured at the reaction site on the insoluble carrier; and a silver amplification step in which the gold particle complex captured in the capture step is silver-amplified. For reasons that provide better effects of the present invention, it is preferable that at least one of the first binding substance and the second binding substance is a monoclonal antibody, and it is preferable that both the first binding substance and the second binding substance are monoclonal antibodies.

[0036] Each step of the preferred embodiment will be described below.

[0037] [Development step] The development step is a step in which a gold particle complex is formed, which is a complex between the antigen in the antigen concentrate obtained in the concentration step described above and modified gold particles, which are gold particles modified with a first binding substance capable of binding to the antigen, and the gold particle complex is developed on an insoluble carrier having a reaction site at which a second binding substance capable of binding to the antigen is immobilized.

[0038] [Gold particle complex] As described above, in the development step, first, a gold particle complex is formed, which is a complex between the antigen in the antigen concentrate obtained in the concentration step described above and modified gold particles, which are gold particles modified with a first binding substance capable of binding to the antigen.

[0039] <Modified Gold Particles> Modified gold particles are gold particles modified with a first binding substance capable of binding to the antigen.

[0040] (Gold Particles) There are no particular limitations on the gold particles, which act as a catalyst for reducing silver ions in the silver amplification step described below.

[0041] The particle size of the gold particles is preferably 100 nm or less, more preferably 50 nm or less, even more preferably 30 nm or less, and particularly preferably 15 nm or less, because the effects of the present invention are more excellent. There is no particular lower limit to the particle size of the gold particles, but because the effects of the present invention are more excellent, the particle size is preferably 1 nm or more, more preferably 2 nm or more, and even more preferably 5 nm or more.

[0042] The particle size can be measured using a commercially available particle size distribution analyzer or the like. Known methods for measuring particle size distribution include optical microscopy, confocal laser microscopy, electron microscopy, atomic force microscopy, static light scattering, laser diffraction, dynamic light scattering, centrifugal sedimentation, electric pulse measurement, chromatography, and ultrasonic attenuation, and devices corresponding to each principle are commercially available. Dynamic light scattering is a preferred method for measuring particle size because of its particle size range and ease of measurement. Commercially available measuring devices using dynamic light scattering include the Nanotrac UPA (Nikkiso Co., Ltd.), the dynamic light scattering particle size distribution analyzer LB-550 (Horiba, Ltd.), and the concentrated particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.). In the present invention, the median diameter (d=50) measured at a measurement temperature of 25°C is used.

[0043] (First Binding Substance) The first binding substance is not particularly limited as long as it can bind to the antigen. However, for reasons of superior effects of the present invention, a protein is preferred, an antibody (e.g., a polyclonal antibody or a monoclonal antibody) is more preferred, and a monoclonal antibody is even more preferred from the viewpoint of achieving higher detection sensitivity. The antibody is not particularly limited, but for example, antisera prepared from the serum of an animal immunized with the antigen or immunoglobulin fractions purified from the antisera can be used. Furthermore, monoclonal antibodies obtained by cell fusion using spleen cells of an animal immunized with the antigen, or fragments thereof [e.g., F(ab')2, Fab, Fab', or Fv] can also be used. These antibodies can be prepared by conventional methods.

[0044] When the antigen is LAM, an example of the first binding substance is the A194-01 antibody described in WO 2017 / 139153. The entire contents of WO 2017 / 139153 regarding the A194-01 antibody are incorporated herein by reference. Another example of the first binding substance when the antigen is LAM is an antibody having the sequence described as MoAb1 in paragraph

[0080] of WO 2013 / 129634. The entire contents of WO 2013 / 129634 regarding the MoAb1 antibody are incorporated herein by reference.

[0045] (Method for Producing Modified Gold Particles) The method for producing the modified gold particles is not particularly limited, and known methods can be used. For example, a chemical bonding method utilizing the chemical bonding between gold and an SH group includes introducing an SH group into an antibody, and then immobilizing the antibody via an Au-S bond formed on the Au surface when the antibody approaches the gold particle due to cleavage of the SH bond.

[0046] [Insoluble Carrier] The insoluble carrier has a reaction site (test line) to which a second binding substance capable of binding to the antigen is immobilized. The insoluble carrier may have multiple test lines according to the type of antigen (e.g., a test line for influenza A virus and a test line for influenza B virus). The insoluble carrier may also have a control line downstream of the test line to confirm the development of the gold particle complex. Furthermore, when a reducing agent solution is used in the silver amplification step described below, the insoluble carrier may also have a color-developing reagent immobilized line downstream of the test line to detect the reducing agent solution. A specific embodiment of the insoluble carrier is, for example, a nitrocellulose membrane 100 having, from upstream, a gold colloid-retaining pad 1, a test line 2, a control line 3, and a color-developing reagent immobilized line 4, as shown in FIG. 13 . Here, gold colloid holding pad 1 is a pad that holds gold particles modified with a first binding substance (modified gold particles), test line 2 is a line where a second binding substance is immobilized, control line 3 is a line for confirming development, and color-developing reagent immobilized line 4 is a line for detecting a reducing agent solution, which will be described later. Here, the terms "upstream" and "downstream" refer to the development of the gold particle complex from the upstream side toward the downstream side. More specific examples of the insoluble carrier (or immunochromatography kit having the same) include the insoluble carrier and immunochromatography kit described in Japanese Patent No. 5728453. The contents of Japanese Patent No. 5728453 regarding the insoluble carrier and immunochromatography kit are all incorporated herein by reference.

[0047] <Insoluble Carrier> The insoluble carrier is preferably a porous carrier. In particular, because the effects of the present invention are more excellent, a nitrocellulose membrane, a cellulose membrane, an acetylcellulose membrane, a polysulfone membrane, a polyethersulfone membrane, a nylon membrane, glass fiber, a nonwoven fabric, a cloth, or a thread is preferred, and a nitrocellulose membrane is more preferred.

[0048] <Second Binding Substance> The second binding substance is not particularly limited as long as it can bind to the antigen. Specific examples and preferred aspects of the second binding substance are the same as those of the first binding substance described above.

[0049] The second binding substance may be the same as or different from the first binding substance described above; however, in order to achieve better effects of the present invention, it is preferable that the second binding substance and the second binding substance are different substances. Furthermore, when the first binding substance and the second binding substance are antibodies, it is preferable that the antibody serving as the first binding substance and the antibody serving as the second binding substance are different from each other in order to achieve better effects of the present invention. Furthermore, when the first binding substance and the second binding substance are antibodies, it is preferable that the epitope of the first binding substance (a portion of the antigen recognized by the first binding substance) and the epitope of the second binding substance (a portion of the antigen recognized by the second binding substance) are different from each other in order to achieve better effects of the present invention. The fact that the epitopes of the antibodies are different can be confirmed, for example, by ELISA (Enzyme-Linked Immunosorbent Assay).

[0050] [Development] There are no particular limitations on the method for developing the formed gold particle complex onto an insoluble carrier having a test line. For example, a method may be used in which a nitrocellulose membrane 100 (or an immunochromatography kit having the same) as shown in Figure 13 described above is prepared, the concentrated antigen solution obtained in the concentration step described above is dripped onto a gold colloid retention pad, and the antigen is moved from the upstream side to the downstream side by capillary action as shown in Figure 13.

[0051] [Capture step] The capture step is a step of capturing the gold particle conjugate at the reaction site of the insoluble carrier. As described above, a second binding substance capable of binding to an antigen is immobilized at the reaction site of the insoluble carrier. Therefore, the gold particle conjugate (complex of antigen and modified gold particle) developed on the insoluble carrier in the development step is captured at the reaction site (test line) of the insoluble carrier. Note that if the sample liquid does not contain an antigen, the gold particle conjugate is not formed and therefore is not captured at the reaction site of the insoluble carrier.

[0052] [Silver Amplification Step] The silver amplification step is a step of amplifying the silver in the gold particle complex captured in the capture step. The silver amplification step is a step of adding silver ions to the insoluble carrier after the capture step, thereby forming large silver particles in the gold particle complex captured at the reaction sites of the insoluble carrier. More specifically, this step involves reducing the silver ions using the gold particles of the gold particle complex as a catalyst to form silver particles (e.g., with a diameter of 10 μm or more). This significantly improves the detection sensitivity of the captured gold particle complex.

[0053] [Preferred embodiment] The method for providing silver ions to the insoluble carrier after the capture step is not particularly limited, but a method using the following reducing agent solution and silver amplification solution is preferred because it provides better effects of the present invention. In addition to the reducing agent solution and silver amplification solution, a washing solution may be used to wash away complexes remaining on the insoluble carrier other than those resulting from the specific binding reaction. The reducing solution may also serve as the washing solution.

[0054] <Reducing Agent Solution> The reducing agent solution contains a reducing agent capable of reducing silver ions. The reducing agent capable of reducing silver ions can be any inorganic or organic material or a mixture thereof, as long as it can reduce silver ions to silver. Examples of inorganic reducing agents include Fe ２＋ , V ２＋ , Ti ３＋ Preferred examples of reducing metal salts and reducing metal complex salts are those whose valence can be changed by metal ions such as Fe, FeN, FeO ... ２＋ In the system using citric acid or ethylenediaminetetraacetic acid (EDTA) as a reducing agent, the oxide Fe ３＋ In the present invention, it is preferable to use such an inorganic reducing agent, and in a more preferred embodiment of the present invention, Fe ２＋ It is preferred to use a metal salt of the above as the reducing agent.

[0055] Developing agents used in wet silver halide photographic light-sensitive materials (e.g., methyl gallate, hydroquinone, substituted hydroquinone, 3-pyrazolidones, p-aminophenols, p-phenylenediamines, hindered phenols, amidoximes, azines, catechols, pyrogallols, ascorbic acid (or its derivatives), and leuco dyes), and other materials obvious to those skilled in the art, such as those described in U.S. Pat. No. 6,020,117, can also be used as reducing agents.

[0056] As a reducing agent, an ascorbic acid reducing agent is also preferred. Useful ascorbic acid reducing agents include ascorbic acid and its analogs, isomers, and derivatives. Preferred examples include D- or L-ascorbic acid and its sugar derivatives (e.g., γ-lactoascorbic acid, glucoascorbic acid, fucoascorbic acid, glucoheptoascorbic acid, and maltoascorbic acid), sodium ascorbic acid, potassium ascorbic acid, isoascorbic acid (or L-erythroascorbic acid), salts thereof (e.g., alkali metal salts, ammonium salts, or salts known in the art), enediol-type ascorbic acid, enaminol-type ascorbic acid, and thioenol-type ascorbic acid. D, L, or D,L-ascorbic acid (and its alkali metal salts) or isoascorbic acid (or its alkali metal salts) are particularly preferred, with sodium salts being the preferred salts. Mixtures of these reducing agents can be used if necessary.

[0057] For reasons why the effects of the present invention are more excellent, the reducing agent solution is preferably flowed so that the angle between the developing direction in the developing step and the developing direction of the reducing agent solution is 0 to 150 degrees, and more preferably so that the angle between the developing direction in the developing step and the developing direction of the reducing agent solution is 0 to 135 degrees. Note that examples of methods for adjusting the angle between the developing direction in the developing step and the developing direction of the reducing agent solution include the methods described in the examples of JP 2009-150869 A.

[0058] <Silver Amplification Solution> The silver amplification solution is a solution containing a compound containing silver ions. Examples of compounds that can be used include organic silver salts, inorganic silver salts, and silver complexes. Preferred examples of silver ion-containing compounds that are highly soluble in solvents such as water include silver nitrate, silver acetate, silver lactate, silver butyrate, and silver thiosulfate. Silver nitrate is particularly preferred. Preferred examples of silver complexes include silver complexes coordinated with a ligand having a water-soluble group such as a hydroxyl group or a sulfonic acid group, such as silver hydroxythioether.

[0059] The organic silver salt, inorganic silver salt, or silver complex is preferably contained in the silver amplification solution at a concentration of 0.001 mol / L to 5 mol / L, more preferably 0.005 mol / L to 3 mol / L, and even more preferably 0.01 mol / L to 1 mol / L, in terms of silver.

[0060] Examples of auxiliary agents for the silver amplification solution include buffers, preservatives, such as antioxidants or organic stabilizers, and speed regulators. Examples of buffers that can be used include buffers using acetic acid, citric acid, sodium hydroxide, or one of their salts, or tris(hydroxymethyl)aminomethane, as well as other buffers commonly used in chemical experiments. By appropriately using these buffers, the amplification solution can be adjusted to an optimal pH. Furthermore, alkylamines can be used as auxiliary antifogging agents, with dodecylamine being particularly preferred. Furthermore, surfactants can be used to improve the solubility of these auxiliary agents, with C being particularly preferred. ９ H １９ -C ６ H ４ -O-(CH ２ CH ２ O) ５０ It's H.

[0061] For reasons why the effects of the present invention are more excellent, the silver amplification liquid is preferably flowed in the opposite direction to that of the developing step described above, and more preferably flowed so that the angle between the developing direction in the developing step and the developing direction of the reducing agent liquid is 45 degrees to 180 degrees. Note that examples of methods for adjusting the angle between the developing direction in the developing step and the developing direction of the silver amplification liquid include the methods described in the examples of JP 2009-150869 A.

[0062] [2] Concentration Jig In the concentration step described above, it is preferable to use the following concentration jig (hereinafter also referred to as the "concentration jig of the present invention") because the effects of the present invention are more excellent.

[0063] The concentration device of the present invention is a concentration device used for concentration processing in a test (e.g., an immunological test) that detects macromolecules (e.g., the above-mentioned antigens) contained in a liquid sample (e.g., the above-mentioned specimen liquid), and comprises: a water-absorbent polymer (highly water-absorbent polymer) for absorbing at least a portion of the water in the liquid sample; and a container, at least a portion of which is soft, that contains the water-absorbent polymer and into which the liquid sample is taken, and is capable of pressing the water-absorbent polymer through the inner wall of the container.

[0064] The concentration device of the present invention is a device used for concentrating a liquid sample in a test such as immunochromatography for detecting a macromolecule contained in the liquid sample.

[0065] The concentration device of the present invention will be described below with reference to the drawings.

[0066] Fig. 1 is a diagram conceptually showing an example of the concentration device of the present invention, and Figs. 2 to 4 are diagrams for explaining the function of the concentration device of the present invention.

[0067] 1 includes a water-absorbent polymer 230 and a container 214 that accommodates the water-absorbent polymer 230. Although not shown, the container 214 includes an intake portion for taking in a liquid sample (hereinafter also referred to as a specimen liquid) and an exhaust portion for discharging a concentrated liquid sample (for example, the above-mentioned antigen concentrate) (hereinafter also simply referred to as a "concentrated liquid"). The specific configuration of the container 214 will be described later.

[0068] The water-absorbing polymer 230 is a superabsorbent polymer (SAP) that has high water absorption. As shown in Fig. 1, the water-absorbing polymer 230 is in a particulate form, and a large number of particulate water-absorbing polymers 230 are contained in the container 214. The superabsorbent polymer is as described above.

[0069] As shown in Fig. 2, when a liquid sample 240 is poured into a container 214 of the concentration device 200 containing a water-absorbent polymer 230 before water absorption, the water-absorbent polymer 230 absorbs the water contained in the liquid sample 240, and the water-absorbent polymer 232 swells after water absorption, as shown in Fig. 3. After water absorption, a portion of the liquid sample (concentrated liquid) 241 that was not absorbed by the water-absorbent polymer 232 remains concentrated in the container 214. Furthermore, a specimen liquid concentrate 246, which is a concentrate of the liquid sample 240, may also be produced.

[0070] In the present invention, at least a portion of the container 214 is flexible, and the water-absorbent polymer 232 can be pressed through the inner wall of the container 214. Therefore, as shown in Fig. 4, the concentrated liquid 242 can be taken out by pressing the swollen water-absorbent polymer 232 after absorbing water through the inner wall of the container 214.

[0071] In addition, after the water-absorbing polymer 232 has absorbed water, a small amount of recovery liquid may be added, and then the swollen water-absorbing polymer 232 after absorption may be pressed through the inner wall of the container 214 to extract the concentrated liquid 242 containing the recovery liquid.

[0072] The inventors' research has revealed that with conventional concentration devices using water-absorbent polymers, it can be difficult to remove the concentrated liquid from the concentration device after concentrating a liquid sample. Specifically, a larger amount of water-absorbent polymer is preferable from the perspective of shortening the concentration time, but if the amount of water-absorbent polymer is too large, the amount of concentrated liquid becomes insufficient, making removal difficult. It has also been considered to remove the concentrated liquid by adding a small amount of recovery liquid after concentration. However, if the amount of water-absorbent polymer is too large, the water-absorbent polymer swells and increases in volume after absorbing water, reducing the void space within the container, making it difficult for the recovery liquid to spread around the water-absorbent polymer, making it difficult to properly recover the sample liquid concentrate remaining near the water-absorbent polymer. Furthermore, if the amount of water-absorbent polymer is too large, water absorption may occur even during the removal operation of the concentrated liquid, potentially preventing a sufficient recovery volume. On the other hand, reducing the amount of water-absorbent polymer or increasing the amount of recovery liquid to ensure a sufficient amount of concentrated liquid results in a low concentration ratio of the concentrated liquid.

[0073] In contrast, in the concentration device 200 of the present invention, at least a portion of the container 214 is flexible, and the absorbent polymer 232 can be pressed through the inner wall of the container 214. Therefore, pressing the absorbent polymer 232 through the inner wall of the container 214 deforms the absorbent polymer 232, reducing the internal volume of the container 214. This creates a stirring effect, allowing a portion of the liquid sample (concentrated liquid 241) that was not absorbed and / or the recovered liquid to spread into the gaps between the absorbent polymers 232. This allows more of the concentrated liquid 241 and specimen liquid concentrate 246 remaining near the absorbent polymer 232 to be recovered, thereby increasing the concentration rate of the concentrated liquid 242. Furthermore, because the concentration device 200 of the present invention can press the absorbent polymer 232 through the inner wall of the container 214, it can directly push the contents (concentrated liquid 241) toward the discharge port. Therefore, the concentrated liquid can be more easily extracted than in a configuration in which the concentrated liquid is pushed out solely by air pressure from a pump or the like. Furthermore, since the concentrated liquid can be efficiently developed and extracted even with a small amount of concentrated liquid, the concentration ratio of the concentrated liquid 242 can be increased. This also makes it easier to ensure the necessary recovery amount of the concentrated liquid 242, and also makes it possible to keep the concentration ratio of the extracted concentrated liquid 242 constant. As a result, variation in the concentration ratio is reduced.

[0074] Furthermore, the concentration device 200 of the present invention can be deformed by pressing the container 214 with the user's fingers, which makes it easy for uneven deformation to occur and allows it to be deformed into various shapes, and since the water-absorbent polymers 232 are movable, it is possible to prevent the water-absorbent polymers 232 from being pressed, narrowing the gaps between the water-absorbent polymers 232 and preventing the concentrated liquid from moving. Therefore, even if the amount of water-absorbent polymers 232 is large, the concentrated liquid can be efficiently spread and extracted.

[0075] Furthermore, the concentration device 200 of the present invention allows the user to remove the concentrated liquid by simply pressing the container 214 with their finger, making the removal operation easy and reducing the time required for the recovery operation.

[0076] As described above, the concentration device 200 of the present invention takes in the liquid sample 240 into the container 214 and discharges the concentrated liquid 242. Therefore, the container 214 has an inlet for taking in the liquid sample 240 and an outlet for discharging the concentrated liquid 242. The inlet is not particularly limited, and various configurations can be used as long as it can take in the liquid sample 240 into the container 214. Similarly, the outlet is not particularly limited, and various configurations can be used as long as it can discharge the concentrated liquid 242 from the container 214. Furthermore, the inlet and outlet may be a common device. However, the inlet preferably has a relatively large opening so that the liquid sample 240 can be easily taken into the container 214 and the water-absorbent polymer before water absorption can be placed in the container 214, and the opening is preferably larger than the particle diameter of the water-absorbent polymer before water absorption. On the other hand, it is preferable that the discharge portion be a relatively large opening that is smaller than the particle diameter of the absorbent polymer after absorption, in order to enable the concentrated liquid to be discharged without discharging the absorbent polymer after absorption, and to prevent air from leaking and making it difficult to remove the concentrated liquid 242 when the container 214 is pressed to discharge the concentrated liquid 242.

[0077] The specific configuration of the container will be described below with reference to the drawings.

[0078] [Container] Fig. 5 is a perspective view schematically showing an example of the concentration device of the present invention, and Fig. 6 is an exploded perspective view of the container shown in Fig. 5.

[0079] The container 214a shown in FIGS. 5 and 6 has a container body 210a, at least a portion of which is flexible and has an opening 216, and a cap 212 that is detachably provided on the opening 216 of the container body 210a.

[0080] 5 and 6, the container body 210a of the container 214a comprises a storage section 211a for storing a water-absorbent polymer and a neck section 215 having an opening 216. In the illustrated example, the storage section 211a is substantially cylindrical in shape with a bottom surface and forms an internal space capable of storing the water-absorbent polymer. The neck section 215 is connected to one of the bottom surfaces, and the opening 216 of the neck section 215 communicates with the internal space of the storage section 211a. In the example shown in FIGS. 5 and 6, the storage section 211a has a tapered section 217a at the end on the neck section 215 side, the diameter of which tapers toward the neck section 215.

[0081] The neck portion 215 is a generally cylindrical portion having an opening 216 penetrating from one bottom surface to the other. In the illustrated example, the neck portion 215 is disposed so that its central axis (the central axis of the cylinder) substantially coincides with the central axis (the central axis of the cylinder) of the storage portion 211a. The neck portion 215 also has a male thread on its outer circumferential surface.

[0082] The area of ​​storage portion 211a in a cross section perpendicular to the central axis is larger than the area of ​​neck portion 215. In the illustrated example, the diameter of storage portion 211a in a cross section perpendicular to the central axis is larger than the diameter of neck portion 215. Therefore, the area of ​​storage portion 211a at the connection position between storage portion 211a and neck portion 215 is larger than the area of ​​neck portion 215. Hereinafter, the bottom surface of storage portion 211a to which neck portion 215 is connected is also referred to as the shoulder portion.

[0083] Furthermore, at least a portion of the storage portion 211a is flexible, and the water-absorbing polymer stored in the storage portion 211a can be pressed via the inner wall of the storage portion 211a. In the illustrated example, it is preferable that at least a portion of the circumferential surface of the storage portion 211a is flexible, and the entire storage portion 211a may be flexible.

[0084] In the example shown in Figures 5 and 6, the cap 212 is a member that closes the opening 216 of the neck portion 215 of the container body 210a. Figure 7 shows a cross-sectional view of the cap 212. As shown in Figures 5, 6, and 7, the cap 212 is a cylindrical member that has one bottom surface and is provided with a female thread on its inner circumferential surface. The cap 212 can be attached to and detached from the container body 210 by threading this female thread into the male thread of the neck portion 215 of the container body 210.

[0085] The cap 212 also has a nozzle 213 that protrudes outward from the bottom surface, and a through-hole that passes through the nozzle 213 is provided, and this through-hole serves as a discharge portion 219 .

[0086] 7, in a preferred embodiment, a filter 220 is disposed on the bottom side inside the cap 212. The filter 220 allows the concentrated liquid to pass through but does not allow the water-absorbing polymer to pass through.

[0087] In the container 214a having such a container body 210a and cap 212, the cap 212 can be removed from the container body 210a, and the water-absorbent polymer 230 before absorbing water can be placed in the storage portion 211a through the opening 216 of the neck portion 215. Also, a liquid sample 240 can be placed through the opening 216 of the neck portion 215. In other words, the opening 216 of the container body 210a is an intake portion for taking in a liquid sample.

[0088] After the liquid sample is taken in, the cap 212 is attached. After a predetermined time has passed, the water-absorbent polymer absorbs the moisture in the liquid sample, concentrating the liquid sample. Thereafter, the concentrated liquid may be stirred in the container 214a as needed, and the concentrated liquid may be discharged from the discharge portion 219 provided on the nozzle 213 of the cap 212. At this time, since at least a portion of the container body 210a is soft and the water-absorbent polymer can be pressed via the inner wall of the container body 210a, the concentrated liquid can be removed by pressing the swollen water-absorbent polymer after absorbing water via the inner wall of the container body 210a.

[0089] Alternatively, after the absorbent polymer has absorbed the water content of the liquid sample, recovery liquid may be added through the opening 216 of the neck 215, and then the cap 212 may be attached. If necessary, the recovery liquid may be stirred in the container 214a, and the concentrated liquid may be removed from the discharge port 219 provided on the nozzle 213 of the cap 212.

[0090] In addition, when stirring the concentrated liquid and / or recovered liquid, a cap without a nozzle 213 (discharge portion 219) may be used to seal the inside of container 214a and stir the liquid, and then when discharging the concentrated liquid, the cap may be replaced with cap 212 having a nozzle 213 to remove the concentrated liquid.

[0091] 5 and 6, the area of ​​the storage portion 211a at the connection position between the storage portion 211a and the neck portion 215 is larger than the area of ​​the neck portion 215, and the storage portion 211a has a shoulder, but this is not limited to this. For example, as shown in the example of Fig. 8, the end of the storage portion 211b of the container body 210b on the neck portion 215 side may have a reduced diameter portion 217b that reduces in diameter toward the neck portion 215 to the same diameter as the neck portion 215, and be connected to the neck portion 215. That is, the example shown in Fig. 8 is an example in which the area (diameter) of the storage portion 211b at the connection position between the storage portion 211b and the neck portion 215 is equal to the area (diameter) of the neck portion 215, and the storage portion 211b does not have a shoulder.

[0092] In addition, in order to prevent deformation of neck portion 215 to which cap 212 is attached when storage portion 211a is pressed to extract the concentrated liquid, it is preferable that storage portion 211a has a shoulder portion, i.e., the area of ​​storage portion 211a at the connection position between storage portion 211a and neck portion 215 is larger than the area of ​​neck portion 215.

[0093] 5 and 6, the end portion on the neck portion 215 side has a reduced diameter portion 217a, but the end portion on the neck portion 215 side does not have to have a reduced diameter portion 217a. The configuration having a reduced diameter portion 217a on the end portion on the neck portion 215 side is preferable because it makes it easier to remove the concentrated liquid.

[0094] 5 and 6, the storage section 211a of the container body 210 has a substantially cylindrical shape, but this is not limiting. For example, the storage section 211a may have a polygonal cylindrical shape such as a triangular cylindrical shape or a square cylindrical shape, or may have an elliptical cylindrical shape.

[0095] Furthermore, the storage section 211a is not limited to a substantially cylindrical shape, and may have various shapes. For example, as shown in the example of Fig. 9, the storage section 211c of the container body 210c may have a substantially circular cross-sectional shape on the neck 215 side and a shape that flattens and reduces in cross-sectional area toward the opposite side from the neck 215.

[0096] 10, the container body 210d may have a neck 215 having an opening 216 to which the cap 212 can be attached or detached, and a bag-like container 218 for containing the water-absorbent polymer. Note that the bag-like container refers to a container made of a material that is not self-supporting.

[0097] Furthermore, in the above example, the container body 210 (210a to 210d) and the cap 212 each have a male thread and a female thread and are configured to be screwed together, but this is not limited to this, and they may be configured to be fixed by various known detachable fixing methods, such as a method of attaching by fitting, or a method of having one side with a convex portion and the other with a concave portion and engaging the concave portion with the convex portion.

[0098] Here, in the present invention, "at least a portion of a container is flexible" means that the flexible portion of the container is made of a resin or elastomer material such as polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS), polypropylene (PP), polyvinyl chloride (PVC), or acrylic resin (PMMA), and has a thickness of 1000 μm or less. Furthermore, in addition to the above materials, the container may be a composite material containing a material that has the necessary functions such as low moisture permeability, gas barrier properties, light blocking properties, and decorative properties. Hereinafter, a container that is at least partially flexible will also be referred to as a "flexible container."

[0099] The resin material of the soft portion of the container is preferably either polyethylene (PE) or polypropylene (PP) from the viewpoints of high flexibility, relatively high strength, chemical resistance, cost, etc.

[0100] The thickness of the flexible portion of the container is preferably 1000 μm or less, more preferably 800 μm or less, even more preferably 600 μm or less, and particularly preferably 400 μm or less, because the effects of the present invention are more excellent. There is no particular lower limit, but the thickness is preferably 20 μm or more, more preferably 100 μm or more, because the effects of the present invention are more excellent.

[0101] In the present invention, it is preferable that the wall surface of the container in the outlet portion, which is parallel to the direction in which the concentrated liquid sample (concentrated liquid) is discharged, is soft. This point will be explained using FIG.

[0102] 11 is a conceptual cross-sectional view of a container 214 included in the concentration device of the present invention. In the container 214 shown in FIG. 11, a discharge part 219 is provided on the upper surface of the container 214 in the drawing. That is, in the illustrated example, the direction in which the concentrated liquid is discharged from the discharge part 219 is upward in the drawing, as indicated by arrow D. Therefore, it is preferable that the wall surface 221 of the container 214 parallel to this arrow D is flexible.

[0103] The container 214 has a flexible wall surface 221 parallel to the direction in which the concentrated liquid is discharged from the discharge portion 219, and this allows the container 214 to be pressed in a direction substantially perpendicular to the direction in which the concentrated liquid is discharged. This prevents the water-absorbent polymer from being pressed, narrowing the gaps between the water-absorbent polymers and preventing the concentrated liquid (recovered liquid) from moving, thereby allowing the concentrated liquid (recovered liquid) to be efficiently spread and facilitating the removal of the concentrated liquid.

[0104] For example, in the example shown in Figure 5, the direction in which the concentrated liquid is discharged is upward in the figure, so it is preferable that at least a portion of the peripheral surface of the storage section 211a of the container body 210a is soft, and it is more preferable that the entire peripheral surface is soft.

[0105] In the present invention, the amount of change in the volume of the container is preferably greater than the difference V-(Vs+Vp) between the volume V of the container and the volume Vs of the liquid sample and the volume Vp of the water-absorbent polymer contained in the container. This point will be explained using FIG. 12.

[0106] Fig. 12 is a diagram conceptually showing the concentration device 200 of the present invention. As shown in Fig. 12, if the total volume of the container 214 is V, the total volume of all the water-absorbent polymers 230 in the container 214 before absorbing water is Vp, and the volume of the liquid sample 240 to be placed in the container 214 is Vs, the difference V-(Vs+Vp) between the volume V of the container, the volume Vp of the water-absorbent polymers 230, and the volume Vs of the liquid sample 240 is the volume Va of the space in the container 214 that is not filled with the water-absorbent polymers 230 and / or the liquid sample 240.

[0107] Since the sum of the volume of the water-absorbent polymer after absorbing water and the volume of the remaining concentrated liquid is approximately equal to the sum of the volume Vp of the water-absorbent polymer 230 before absorbing water and the volume Vs of the liquid sample 240, by making the amount of change in volume of the container 214 larger than the difference V-(Vs+Vp), that is, by making it larger than the volume Va of the space in the container 214 before deformation that is not filled with the water-absorbent polymer 230 and / or liquid sample 240, i.e., the void portion in the container 214, the concentrated liquid can be more reliably discharged when discharging the concentrated liquid from the container 214, even if at least a portion of the air in the container 214 is discharged.

[0108] In the present invention, the total surface area S of the container wall １ The surface area of ​​the flexible wall S ２ It is preferable that the ratio of the total surface area S of the container wall is 50% or more. １ The surface area of ​​the flexible wall S ２ By making the ratio of the volumetric capacity of the container 214 50% or more, the volumetric capacity of the container 214 can be increased, and the concentrated liquid can be discharged more reliably when the concentrated liquid is discharged from the container 214 .

[0109] As in the example shown in FIG. 5 , when the container 214 has a container body 210 and a cap 212, it is preferable that at least a portion of the circumferential surface of the storage portion 211 of the container body 210 is flexible, and it is more preferable that the entire circumferential surface is flexible. The entire storage portion 211 may be flexible. The neck portion 215 and the cap 212 may be either flexible or non-flexible, but non-flexible is preferable. When the storage portion 211 and the neck portion 215 are integrally formed from the same material, the storage portion 211 can be flexible and the neck portion 215 can be non-flexible by making the thicknesses different. The storage portion 211 and the neck portion 215 may also be formed from different materials.

[0110] In the present invention, the distance from the tip of the discharge part of the container to the soft wall surface (i.e., the pressing part) in a direction parallel to the direction in which the concentrated liquid is discharged is preferably 70 mm or less, more preferably 50 mm or less, and even more preferably 30 mm or less. There is no particular lower limit, but it is preferably 1 mm or more. This allows the concentrated liquid to be discharged more reliably when it is discharged from the container.

[0111] In the present invention, the ratio of the width in the direction perpendicular to the height direction (the diameter of the cross section in the case of a cylindrical shape) of the container's storage section that stores the water-absorbent polymer to the height in the direction in which the concentrated liquid is discharged is preferably 8 or less, more preferably 5 or less, and even more preferably 3 or less, and although there is no particular restriction on the lower limit of the ratio, it is preferably 0.5 or more. This allows the concentrated liquid to be discharged more reliably when it is discharged from the container.

[0112] [Superabsorbent Polymer] The superabsorbent polymer (superabsorbent polymer) used in the concentration jig of the present invention is as described above.

[0113] [Binding substance that specifically binds to macromolecules contained in biological fluids] In order to increase detection sensitivity when performing a test using a concentrate obtained using the concentrating device of the present invention, it is preferable that the container further contains a binding substance that specifically binds to macromolecules contained in the biological fluid in the sample liquid, as described below. When the container contains the binding substance, for example, an antigen-antibody reaction proceeds simultaneously with the concentration of the sample liquid, and a complex between the antigen in the sample liquid and the labeled antibody is formed in a concentrated state, leading to improved detection sensitivity.

[0114] The binding substance may be, for example, a first binding substance (particularly an antibody) described below. That is, in the present invention, it is preferable that the macromolecule contained in the biological fluid is an antigen and the binding substance is an antibody.

[0115] The binding substance is preferably contained in the container as a complex with a labeling substance. Examples of the complex include a labeled antibody. Here, the labeled antibody refers to an antibody bound to a detectable labeling substance, and the labeling substance refers to, for example, a detectable substance, such as a substance that can be directly detected, for example, a substance that can generate electromagnetic waves such as color, fluorescence, or light, or a substance that can scatter electromagnetic waves such as color, fluorescence, or light, or a substance or state that includes an enzyme or the like that forms a luminescent or chromogenic body by interacting with a luminescent precursor or a chromogenic precursor.

[0116] The labeled antibody is preferably an antibody modified with metal particles that exhibit a vivid color when irradiated with electromagnetic waves such as visible light. The metal particles are more preferably gold particles. The labeled antibody is preferably an antibody labeled with gold particles, i.e., gold particles modified with an antibody (modified gold particles, described below). The labeled antibody may be contained in a container as a pad (gold colloid-holding pad) holding modified gold colloid particles, which are gold colloid particles modified with an antibody.

[0117] [Casein, Tricine] In view of increasing detection sensitivity when testing using a concentrate concentrated using the concentration device of the present invention, it is preferable that the container further contains at least one selected from the group consisting of casein and tricine, and it is more preferable that the container contains both casein and tricine.

[0118] Casein is thought to have the effect of suppressing false positives. Furthermore, when the pH of a sample liquid, such as urine, is on the acidic side, false positives tend to occur, but tricin is thought to have the effect of suppressing false positives by adjusting the pH to neutral or alkaline.

[0119] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0120] [1] Fabrication of Concentration Jigs Concentration jigs as shown in Figures 5 to 7 were fabricated. The container body 210a was made of polyethylene. The thickness of the storage section 211a was 600 μm. Therefore, the storage section 211a was flexible. The storage section 211a contained AQUALIC CA H2 (swelling ratio: 24 g / g, particle size: 700 μm) manufactured by Nippon Shokubai Co., Ltd. (not shown). Four types of concentration jigs were fabricated by varying the amount of SAP (hereinafter also referred to as "SAP amount") contained in the storage section 211a to 0.5 g, 1.0 g, 1.5 g, and 2.0 g. As described above, the storage section 211a was flexible, allowing the SAP to be pressed through the inner wall of the container.

[0121] [2] Preparation of sample solution Lipoarabinomannan (LAM) extracted from Mycobacterium tuberculosis (product number 02249-61, manufactured by Nacalai Tesque, Inc.) was added to artificial urine (product number 90048945, manufactured by Isekyu Co., Ltd.) to prepare artificial urine (sample solution) containing 25 pg / mL of LAM.

[0122] [3] Preparation of a gold colloid holding pad A solution containing gold colloid particles (particle diameter: 50 nm) (product number: EM.GC50, manufactured by BBI) of 9 mL was diluted with 50 mmol / L of KH ２ P.O. ４The pH was adjusted by adding 1 mL of buffer (pH 8.0). To the pH-adjusted solution, 1 mL of a solution containing 20 μg / mL of anti-lipoarabinomannan (LAM) monoclonal antibody (Nacalai Tesque, Inc., product code 05494-84) was added and stirred for 10 minutes. After leaving it to stand for 10 minutes, 550 μL of an aqueous solution containing 1% by mass of polyethylene glycol (PEG (polyethylene glycol); weight average molecular weight (Mw.): 20000, product number: 168-11285, Fujifilm Wako Pure Chemical Industries, Ltd.) was added and stirred for 10 minutes, followed by 1.1 mL of an aqueous solution of 10% by mass of bovine serum albumin (BSA (Bovine serum albumin); Fraction V, product number: A-7906, SIGMA Corporation) and stirred for 10 minutes. This solution was centrifuged at 8,000 x g and 4°C for 30 minutes using a centrifuge (himacCF16RX, Hitachi, Ltd.). The supernatant was removed, leaving 1 mL at the bottom of the container. The colloidal gold particles contained in the 1 mL of solution remaining at the bottom of the container were redispersed using an ultrasonic cleaner. The particles were then dispersed in 20 mL of colloidal gold storage solution (20 mmol / L Tris-HCl (Tris-HCl) buffer (pH 8.2), 0.05% PEG (Mw. 20,000), 150 mmol / L NaCl, 1% BSA), and centrifuged again using the same centrifuge under the same conditions. The supernatant was removed, ultrasonically dispersed, and then dispersed in colloidal gold storage solution to obtain a solution of antibody-modified colloidal gold particles (labeled antibody), which were colloidal gold particles (particle diameter: 50 nm) modified with anti-LAM monoclonal antibody. The concentration of the obtained solution was adjusted with a buffer, and then the solution was dropped onto a 5 mm x 30 cm glass fiber pad (Merck GFDX203000). The pad was then dried in a vacuum dryer for 15 hours and cut into pieces to obtain pads (gold colloid-retaining pads) (5 mm x 4 mm) that retained antibody-modified gold colloid particles (labeled antibody), which were gold colloid particles modified with an anti-LAM monoclonal antibody.

[0123] [4] Immunochromatography

[0124] [Example 1]

[0125] [Concentration step] 12 mL of the above-mentioned sample solution was added to the concentration device (SAP amount: 0.5 g) prepared as described above and mixed. In this way, the sample solution and SAP were mixed. After standing for 20 minutes, 4 mL of artificial urine containing 0.1% BSA (bovine serum albumin) (product number: A7906, manufactured by SIGMA) was added to the concentration device and stirred for 30 seconds. Then, the SAP was pressed through the inner wall of the soft container of the concentration device to remove the antigen concentrate from the concentration device. After the molecular weight analysis described below, a calibration curve was created from the absorbance measurement of the sample polymer solution, and the polymer concentration was determined. The polymer concentration in the removed antigen concentrate was 10 mg / mL. The polymer contained in the antigen concentrate was the specific polymer described above.

[0126] 13, a nitrocellulose membrane 100 was prepared having, from the upstream side, a gold colloid-retaining pad 1, a test line 2, a control line 3, and a color-developing reagent immobilized line 4. The gold colloid-retaining pad 1 is a pad that retains gold colloids (modified gold particles) modified with an anti-LAM monoclonal antibody, the test line 2 is a line where the anti-LAM monoclonal antibody is immobilized, the control line 3 is a line for confirming development, and the color-developing reagent immobilized line 4 is a line for detecting the reducing solution in the silver amplification step described below.

[0127] The resulting concentrated antigen solution (entire volume) was dropped onto the colloidal gold retention pad 1. This resulted in the formation of a gold particle complex, which was a complex between LAM in the solution and colloidal gold particles (modified gold particles) modified with anti-LAM monoclonal antibodies. The formed gold particle complex was spread from the upstream side to the downstream side of the nitrocellulose membrane.

[0128] [Capturing Step] The gold particle complex developed in the developing step is captured at the test line 2.

[0129] [Silver Amplification Step] The silver amplification step was carried out as follows.

[0130] <Preparation of reducing agent solution> 23.6 mL of a 1 mol / L aqueous solution of iron nitrate, prepared by dissolving iron (III) nitrate nonahydrate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in water, and 13.1 g of citric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were dissolved in 290 g of water. After all the components were dissolved, 36 mL of nitric acid (10% by mass) was added while stirring with a stirrer, and 60.8 g of ammonium iron (II) sulfate hexahydrate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to prepare a reducing agent solution.

[0131] <Preparation of silver amplification solution> 8 mL of silver nitrate solution (containing 10 g of silver nitrate) and 24 mL of 1 mol / L aqueous iron nitrate solution were added to 66 g of water. This solution, 5.9 mL of nitric acid (10% by mass), 0.1 g of dodecylamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and surfactant C were further added. １２ H ２５ -C ６ H ４ -O-(CH ２ CH ２ O) ５０ A solution prepared by dissolving 0.1 g of H in 47.6 g of water was mixed and used as a silver amplification solution.

[0132] <Development of reducing agent solution> The reducing agent solution prepared as described above was allowed to flow through the nitrocellulose membrane in the same direction (more upstream) as in the development step described above.

[0133] <Development of silver amplification solution> After the color-developing reagent immobilized line changed color, the silver amplification solution prepared as described above was flowed in the opposite direction (downstream) to the development direction in the development step. In this way, the gold particle complex captured on test line 2 was silver-amplified.

[0134] 〔evaluation〕

[0135] <BG Darkening> Darkening of the background (coloring of areas other than the test line, control line, and color-developing reagent immobilized line) (hereinafter also referred to as "BG") (hereinafter also referred to as "BG darkening") was visually confirmed, and BG darkening was evaluated according to the following criteria. The results are shown in Table 1. +++: Darkening of the background was very clearly confirmed. ++: Darkening of the background was clearly confirmed. +: Darkening of the background was confirmed. ±: Darkening of the background was barely confirmed. -: Darkening of the background was not confirmed.

[0136] <Sensitivity> The coloration of the test line (difference from the background) was confirmed visually, and the sensitivity was evaluated according to the following criteria. The results are shown in Table 1. +++: The coloration of the test line was extremely clearly confirmed. ++: The coloration of the test line was clearly confirmed. +: The coloration of the test line was confirmed. ±: The coloration of the test line was barely confirmed. -: The coloration of the test line was not confirmed.

[0137] <False Positives> Immunochromatography (concentration step to silver amplification step) was performed as described above, except that artificial urine without added LAM was used instead of the sample liquid. Then, the coloration of the test line (difference from background) was visually confirmed, and false positives were evaluated according to the following criteria. The results are shown in Table 1. +: Some coloration of the test line was observed (false positive). -: No coloration of the test line was observed.

[0138] [Example 2] Immunochromatography was performed and each evaluation was carried out according to the same procedure as in Example 1, except that a concentration device (SAP amount: 1.0 g) was used as the concentration device. The results are shown in Table 1. The polymer concentration in the antigen concentrate was 20 mg / mL. The polymer contained in the antigen concentrate was the specific polymer described above.

[0139] [Example 3] Immunochromatography was performed and each evaluation was carried out according to the same procedure as in Example 1, except that a concentration device (SAP amount: 1.5 g) was used as the concentration device. The results are shown in Table 1. The polymer concentration in the antigen concentrate was 30 mg / mL. The polymer contained in the antigen concentrate was the specific polymer described above.

[0140] Comparative Example 1 Immunochromatography was performed and each evaluation was carried out according to the same procedure as in Example 1, except that a concentration device (SAP amount: 2.0 g) was used as the concentration device. The results are shown in Table 1. The polymer concentration in the antigen concentrate was 40 mg / mL. The polymer contained in the antigen concentrate was the specific polymer described above.

[0141] Comparative Example 2 Immunochromatography was performed and each evaluation was carried out in the same manner as in Example 1, except that the concentration step was not performed and a sample solution was used instead of the antigen concentrate. The results are shown in Table 1. The polymer concentration in the sample solution was 0 mg / mL.

[0142]

[0143] In Table 1, the column "SAP [g]" indicates the SAP amount [g] of the concentration jig. In Table 1, the column "Polymer concentration [mg / mL]" indicates the polymer concentration [mg / mL] in the antigen concentrate (sample solution in Comparative Example 2).

[0144] As can be seen from Table 1, compared to Comparative Example 1, in which the polymer concentration in the antigen concentrate was 35 mg / mL or more, and Comparative Example 2, in which the polymer concentration in the sample solution was 0 mg / mL, Examples 1 to 3, in which the polymer concentration in the antigen concentrate was greater than 0 mg / mL and less than 35 mg / mL, showed excellent sensitivity. Among them, Examples 2 and 3, in which the polymer concentration in the antigen concentrate was 15 mg / mL or more, showed even better sensitivity. Among them, Example 3, in which the polymer concentration in the antigen concentrate was 25 mg / mL or more, showed even better sensitivity. Note that since the amount of LAM in the antigen concentrates in Examples 1 to 3 and Comparative Examples 1 and 2 (sample solution in Comparative Example 2) is almost the same, the difference in sensitivity between Examples 1 to 3 and Comparative Examples 1 and 2 is thought to be due to the difference in polymer concentration in the antigen concentrates (sample solution in Comparative Example 2).

[0145] Furthermore, Comparative Example 1, in which the polymer concentration in the antigen concentrate was 35 mg / mL or more, showed a false positive, whereas Examples 1 to 3, in which the polymer concentration in the antigen concentrate was less than 35 mg / mL, did not show a false positive.

[0146] [5] Analysis of residual polymer in concentrated solution

[0147] [Concentration Step] Commercially available superabsorbent polymer (SAP) particles A (product number: SAP Spheres, manufactured by M2 Polymer Technologies) (lots #1 and #2) and particles B (product number: polymerSNOW, manufactured by M2 Polymer Technologies) (#3) were used as SAP in the concentration step. 12 mL of ultrapure water and the SAP were added to the concentration jig prepared as described above and mixed. After standing for 20 minutes, 4 mL of ultrapure water was added and stirred, and the concentrate was removed from the concentration jig.

[0148] [Structural analysis] The above concentrated solution was centrifuged, and the supernatant was １ H-NMR spectroscopy was performed. As shown in Figure 14, peak 3 (circled number in Figure 14) derived from the acrylic main chain and peaks 1 and 2 (circled numbers in Figure 14) derived from the amide groups were detected in #1 and #2, and the integral values ​​of the acrylic main chain and amide groups were 3.0:1.7 (#1) and 3.0:1.6 (#2). In the case of a polyacrylamide homopolymer, the proton ratio of the acrylic main chain to the amide groups is 3:2, so it was estimated that this measurement also contained acrylic acid, whose side chains could not be detected. From the above, #1 and #2 were estimated to be copolymers of acrylic acid and acrylamide. Furthermore, peaks derived from the acrylic main chain and amide groups were not detected in #3.

[0149] [Molecular Weight Analysis] The concentrate was centrifuged, and the supernatant was diluted with 100 mM phosphate buffer (pH 6.8) and analyzed by gel permeation chromatography (GPC). 0.1 wt% sodium polyacrylate (1250, 16000, 82900, 193800, 782200, 1100000) was used as a standard. As shown in Figure 15, Mw = 1.6 × 10 from all eluates #1, #2, and #3. ５~3.1 × 10 ５ The amount of high molecular weight components detected (area ratio) was the highest in #2, and the amount detected in #3 was the smallest.

[0150] [Effect on immunochromatography reaction] Immunochromatography was performed in the same manner as in Example 1, except that the above concentrate was used instead of the antigen concentrate, and BG blackening and false positives were evaluated. The results are shown in Table 2.

[0151]

[0152] As can be seen from Table 2, the higher the polymer content in the concentrate, the darker the background became and the more false positives were observed.

[0153] [6] Evaluation of the effect of polymer addition on immunochromatographic reaction

[0154] [Preparation of polymer solution] 400 mg of sodium polyacrylate (polymerization degree 22,000 to 70,000) (product number: 196-02955, Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 10 mL of water. The solution was diluted two-fold to prepare polymer solutions of various concentrations.

[0155] [Evaluation] Immunochromatography was performed according to the same procedure as in Example 1, except that the above concentrate was used instead of the antigen concentrate. The immunochromatography was detected using an LAS 4000 (manufactured by Fujifilm Corporation), and the obtained image was analyzed using a Multi-gauge to calculate the background value. As shown in Figure 16, an increase in the BG of immunochromatography was confirmed depending on the concentration of the polymer solution.

[0156] 1 gold colloid holding pad 2 test line 3 control line 4 color-developing reagent immobilization line 100 nitrocellulose membrane 200 concentration jig 210, 210a, 210b, 210c, 210d container body 211, 211a, 211b, 211c storage section 212 cap 213 nozzle 214, 214a container 215 neck 216 opening 217a, 217b diameter-reduced section 218 bag-shaped storage section 219 discharge section 220 filter 221 wall surface 230 water-absorbent polymer (water-absorbent polymer before water absorption) 232 water-absorbent polymer (water-absorbent polymer after water absorption, swollen water-absorbent polymer) 240 liquid sample (analyte liquid) 241 concentrated liquid 242 extracted concentrated liquid 246 sample liquid concentrate

Claims

1. An immunoassay method comprising: a concentration step of concentrating a liquid that may contain an antigen by mixing the liquid with a superabsorbent polymer to obtain an antigen concentrated solution; and a detection step of detecting the antigen in the antigen concentrated solution using an antigen-antibody reaction, wherein the content of polymers having a weight-average molecular weight of less than 1,000,000 in the antigen concentrated solution is more than 0 mg / mL and less than 35 mg / mL.

2. The immunoassay method according to claim 1, wherein the highly water-absorbent polymer is a polyacrylic acid-based or polyacrylamide-based polymer.

3. The immunoassay method according to claim 1 or 2, wherein the polymer is at least one polymer or copolymer selected from the group consisting of acrylic acid, sodium acrylate, and acrylic acid amide.

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