Method for concentrating a test body fluid, and method for testing a test body fluid

Abstract

Provided is a method for concentrating a test body fluid to obtain a test body fluid concentrate having a desired concentration factor, and a method for testing a test body fluid using the method for concentrating a test body fluid. A method for concentrating a test body fluid sequentially includes: a test body fluid injection step of injecting a test body fluid, which is an aqueous solution containing a high molecule, into a cylinder body in which a particulate super absorbent polymer is accommodated; a water absorption step of absorbing water contained in the test body fluid injected into the cylinder body by the super absorbent polymer accommodated in the cylinder body to generate a test body fluid concentrate, which is a concentrate of the test body fluid, in the cylinder body; a liquid addition step of adding a liquid to the test body fluid concentrate, the liquid being less than the test body fluid injected into the cylinder body in the test body fluid injection step; and a removal step of inserting a piston, which is capable of being inserted into the cylinder body and has a front end portion having a hole smaller than a particle size of the super absorbent polymer after water absorption, into the cylinder body to remove a test body fluid concentrate, which is a concentrate liquid of the test body fluid, through the hole of the front end portion of the piston.

Description

Technical Field

[0001] This invention relates to a method for concentrating a body fluid sample and a method for examining the body fluid sample. Background Technology

[0002] Previously, techniques were known to concentrate aqueous solutions containing high molecules such as antigens (hereinafter also referred to as "test fluid") using superabsorbent polymers (e.g., Patent Document 1).

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Application Publication No. 4-355339 Summary of the Invention

[0006] The technical problem to be solved by the invention

[0007] In this context, the inventors, referring to Patent Document 1, etc., studied a method for concentrating test fluids using superabsorbent polymers. They found that it was difficult to control the concentration ratio of the concentrated test fluid (test fluid concentrate). When using a test fluid concentrate with an uncontrolled concentration ratio for testing, problems arise because comparisons between the test fluids are hindered.

[0008] Therefore, in view of the above, the object of the present invention is to provide a method for concentrating a test body fluid that can obtain a concentrated solution of the test body fluid at a desired concentration ratio, and a method for examining the test body fluid using the above-described method for concentrating the test body fluid.

[0009] means for solving technical problems

[0010] The inventors conducted in-depth research on the above-mentioned problems and found that by injecting the test fluid into a cylinder containing a superabsorbent polymer, the water in the test fluid is almost completely absorbed by the superabsorbent polymer to generate a concentrate of the test fluid. Then, liquid is added to it, and the liquid is removed using a perforated piston. This solves the above-mentioned problems and completes the present invention.

[0011] That is, the inventors have discovered that the above-mentioned problems can be solved by the following structure.

[0012] (1) A method for concentrating a body fluid sample, comprising, in sequence:

[0013] The test fluid injection process involves injecting the test fluid, which is an aqueous solution containing a high molecular weight polymer, into a cylinder containing particulate superabsorbent polymer.

[0014] In the water absorption process, the water contained in the test fluid injected into the above-mentioned cylinder is absorbed by the superabsorbent polymer contained in the above-mentioned cylinder, and a test fluid concentrate, which is a concentrate of the test fluid, is generated in the above-mentioned cylinder.

[0015] In the liquid addition step, a smaller amount of liquid than the amount of test body fluid injected into the cylinder during the test body fluid injection step is added to the above-mentioned test body fluid concentrate; and

[0016] In the removal process, the piston is inserted into the cylinder, thereby removing the concentrated body fluid of the test fluid, which is the concentrated body fluid of the test fluid, through the hole at the front end of the piston. The piston can be inserted into the cylinder and has a front end with a hole that is smaller than the particle size of the superabsorbent polymer after water absorption.

[0017] (2) According to the concentration method of the tested body fluid described in (1) above, wherein,

[0018] The aforementioned test fluid injection process involves injecting the test fluid into the aforementioned cylinder while simultaneously maintaining a portion of the injected test fluid within the cylinder as part of the liquid added during the aforementioned liquid addition process.

[0019] The water absorption process described above involves the absorption of water contained in the test fluid (excluding the test fluid held in place by the liquid added in the liquid addition process) injected into the cylinder by a highly absorbent polymer contained in the cylinder, thereby generating a concentrate of the test fluid in the cylinder.

[0020] The above-mentioned liquid addition process is a process of adding the test body fluid as a liquid to the above-mentioned test body fluid concentrate, which is used to maintain the test body fluid added in the above-mentioned liquid addition process.

[0021] (3) The concentration method for the tested body fluid as described in (2) above, wherein,

[0022] The cylinder has a liquid holding portion at its bottom for holding the liquid added in the liquid adding process. The superabsorbent polymer is contained in the cylinder above and in contact with the liquid holding portion.

[0023] The above-mentioned test fluid injection process is a process in which the test fluid is injected into the cylinder, and a portion of the test fluid injected into the cylinder is retained in the liquid holding section as liquid added in the above-mentioned liquid adding process.

[0024] (4) The concentration method for the tested body fluid as described in (3) above, wherein,

[0025] The liquid holding section is the portion surrounded by the bottom of the cylinder and a partition wall. The partition wall is disposed on the inner circumferential surface of the cylinder in a manner that allows it to move along the length of the cylinder. The partition wall has pores smaller than the particle size of the superabsorbent polymer before water absorption.

[0026] The above-mentioned liquid addition process involves moving the partition wall to the bottom surface of the cylinder, introducing the test body liquid held in the liquid holding section through the hole of the partition wall to the top of the partition wall, thereby adding the test body liquid held in the liquid holding section to the test body liquid concentrate.

[0027] (5) The concentration method for the tested body fluid as described in (3) above, wherein,

[0028] The liquid holding section is formed by the pores of a porous resin housed at the bottom of the cylinder, wherein the pores of the resin are smaller than the particle size of the superabsorbent polymer before water absorption.

[0029] The above-mentioned liquid addition process is a process of crushing the resin and introducing the test body fluid held in the liquid holding section through the pores of the resin to the top of the resin, thereby adding the test body fluid held in the liquid holding section to the test body fluid concentrate.

[0030] (6) The method for concentrating the tested body fluid according to (5) above, wherein the porous resin is a sponge.

[0031] (7) The concentration method for the tested body fluid as described in (2) above, wherein,

[0032] The aforementioned test fluid injection process involves simultaneously injecting the test fluid into the cylinder, inserting the piston into the cylinder, and fixing the front end of the piston at a position lower than the liquid level of the test fluid injected into the cylinder but higher than the superabsorbent polymer contained in the cylinder. This process maintains the portion of the test fluid injected into the cylinder above the front end of the piston within the cylinder as liquid added in the aforementioned liquid addition process.

[0033] The above-mentioned liquid addition process is to pull up the piston and introduce the test fluid present above the front end of the piston through the hole at the front end of the piston to the area below the front end of the piston, thereby adding the test fluid present above the front end of the piston to the test fluid concentrate.

[0034] (8) The method for concentrating the test body fluid according to any one of (1) to (7) above, wherein, in the above-mentioned extraction step, a cap is further used to recover the extracted test body fluid concentrate, the cap having a recovery port for recovering the test body fluid concentrate.

[0035] (9) The method for concentrating the body fluid of the test according to any one of (1) to (8) above, wherein the water absorption rate of the superabsorbent polymer is more than 0.01 g / min and less than 40 g / min per 1 g of superabsorbent polymer.

[0036] (10) The method for concentrating the body fluid to be tested according to any one of (1) to (9) above, wherein the particle size of the superabsorbent polymer is 5 mm or less.

[0037] (11) The method for concentrating the body fluid according to any one of (1) to (10) above, wherein the swelling ratio of the superabsorbent polymer is greater than 0.2 g / g and less than 800 g / g.

[0038] (12) The method for concentrating the body fluid to be tested according to any one of (1) to (11) above, wherein the body fluid to be tested is an aqueous solution containing high molecules contained in biological body fluids.

[0039] (13) According to the concentration method of the tested body fluid described in (12) above, wherein the cylinder also contains a binding substance that specifically binds to the high molecular weight contained in the biological body fluid.

[0040] (14) The method for concentrating the tested body fluid according to (13) above, wherein the above-mentioned binding substance is contained in the above-mentioned cylinder as a complex with metal particles.

[0041] (15) The method for concentrating the tested body fluid according to (13) or (14) above, wherein the high molecular weight contained in the above-mentioned biological body fluid is an antigen, and the above-mentioned binding substance is an antibody against the above-mentioned antigen.

[0042] (16) The method for concentrating the body fluid to be tested according to any one of (1) to (15) above, wherein the cylinder further contains at least one of the group consisting of casein and tris(hydroxymethyl)glycine.

[0043] (17) The method for concentrating the body fluid to be tested according to any one of (1) to (16) above, wherein the body fluid to be tested is urine.

[0044] (18) A method for examining a body fluid, wherein the test fluid is an aqueous solution containing macromolecules, the method comprising: a concentration step, wherein a concentrated solution of the test fluid is obtained using the concentration method of any one of (1) to (17) described above; and

[0045] The testing process involves detecting the high molecular weight molecules in the obtained concentrated body fluid solution.

[0046] (19) According to the examination method for the tested body fluids described in (18) above, wherein,

[0047] The body fluids tested were aqueous solutions that may contain antigens.

[0048] The above-described concentration step is a step of concentrating an aqueous solution that may contain the above-described antigens using the concentration method for the tested body fluid described in any one of 1 to 17 above to obtain an antigen concentrate.

[0049] The above-mentioned detection procedure is a process of detecting the antigen in the above-mentioned antigen concentrate by using an immunochromatographic method of antigen-antibody reaction.

[0050] (20) According to the inspection method described in (19) above, wherein the detection process includes an amplification process for amplifying information of the antigen in the antigen concentrate.

[0051] (21) According to the inspection method described in (20) above, wherein the amplification process is a silver amplification process.

[0052] Invention Effects

[0053] As described below, according to the present invention, a method for concentrating a test body fluid that can obtain a concentrated solution of the test body fluid at a desired concentration ratio can be provided, and a method for examining the test body fluid using the above-described method for concentrating the test body fluid can be provided. Attached Figure Description

[0054] Figure 1A This is a schematic cross-sectional view showing the initial state of one way in which the concentration method of the present invention is represented according to the process sequence.

[0055] Figure 1B This is a schematic cross-sectional view showing the injection process of the test body fluid in one embodiment of the concentration method of the present invention, arranged in the order of the processes.

[0056] Figure 1C This is a schematic cross-sectional view showing the water absorption process in one embodiment of the concentration method of the present invention, arranged in the order of the processes.

[0057] Figure 1DThis is a schematic cross-sectional view showing the extract addition process in one embodiment of the concentration method of the present invention, arranged in the order of the processes.

[0058] Figure 1E This is a schematic cross-sectional view showing the extraction process in one embodiment of the concentration method of the present invention, arranged in the order of the processes.

[0059] Figure 2A It is a schematic cross-sectional view showing the initial state in one of the ways of representing the process sequence A1.

[0060] Figure 2B This is a schematic cross-sectional view showing the injection process of the tested body fluid in one of the ways of representing the process sequence, A1.

[0061] Figure 2C This is a schematic cross-sectional view showing the water absorption process in one of the ways of representing the process sequence, A1.

[0062] Figure 2D This is a schematic cross-sectional view showing the extraction liquid addition process in one of the process sequence representation methods A1.

[0063] Figure 2E This is a schematic cross-sectional view showing the extraction process in one of the process sequence representation methods A1.

[0064] Figure 3A It is a schematic cross-sectional view showing the initial state of a method of representing the process sequence A2.

[0065] Figure 3B This is a schematic cross-sectional view showing the injection process of the tested body fluid in one of the ways of representing the process sequence, A2.

[0066] Figure 3C This is a schematic cross-sectional view showing the water absorption process in one of the ways of representing the process sequence, A2.

[0067] Figure 3D This is a schematic cross-sectional view showing the extraction liquid addition process in one of the process sequence representations, A2.

[0068] Figure 3E This is a schematic cross-sectional view showing the extraction process in one of the process sequence representation methods A2.

[0069] Figure 4A It is a schematic cross-sectional view showing the initial state of a method B that represents the process sequence.

[0070] Figure 4BThis is a schematic cross-sectional view showing the injection process of the tested body fluid in one of the ways of representing the process sequence, method B.

[0071] Figure 4C This is a schematic cross-sectional view showing the water absorption process in one of the ways of representing the process sequence, method B.

[0072] Figure 4D This is a schematic cross-sectional view showing the extraction liquid addition process in one of the ways of representing the process sequence, method B.

[0073] Figure 4E This is a schematic cross-sectional view showing the extraction of the process in one of the ways of representing the process sequence, method B.

[0074] Figure 5 This is a schematic cross-sectional view of one embodiment of the condensation device of the present invention.

[0075] Figure 6 This is a schematic cross-sectional view of method A1.

[0076] Figure 7 This is a schematic cross-sectional view of method A2.

[0077] Figure 8 This is a schematic cross-sectional view of method B.

[0078] Figure 9 This is a three-dimensional view of part of the condensation device 201-204.

[0079] Figure 10 This is a three-dimensional view of part of the condenser device 204.

[0080] Figure 11 This is a schematic diagram of one type of insoluble carrier used in the detection step of the inspection method of the present invention.

[0081] Figure 12 This is a three-dimensional diagram showing the form of one embodiment of an immunochromatographic reagent kit.

[0082] Figure 13 This is an exploded schematic perspective view showing the form of one embodiment of an immunochromatographic reagent kit.

[0083] Figure 14 This is a schematic side view showing the positional relationship between the test strips, the first and second cans.

[0084] Figure 15 It is set in Figure 12 A perspective view of the first convex deformable portion on the upper shell of the immunochromatographic reagent kit shown.

[0085] Figure 16 yes Figure 15 The end face view of the V-V' line cut section before and after deformation of the first convex deformed part is shown.

[0086] Figure 17 It is set in Figure 12 A perspective view of the second convex deformable portion on the upper shell of the immunochromatographic reagent kit.

[0087] Figure 18 yes Figure 17 The end face view of the VII-VII' line-cut section before and after deformation of the second convex deformed part is shown.

[0088] Figure 19 This is a cut end face view of the convex deformable part before and after deformation in a design change example. Detailed Implementation

[0089] The following describes the method for concentrating the body fluid to be tested and the method for testing the body fluid to be tested according to the present invention.

[0090] In addition, in this specification, the numerical range indicated by “~” refers to the range including the values ​​recorded before and after “~” as the lower limit and upper limit values.

[0091] Furthermore, in this specification, each ingredient may be used individually or in combination with two or more ingredients. When two or more ingredients are used simultaneously, unless otherwise specified, the content of that ingredient refers to the total content.

[0092] Furthermore, in this specification, the superior effects of the present invention are also referred to as the following: the ability to obtain a concentrated solution of the test body fluid with the desired concentration ratio in the concentration method of the test body fluid of the present invention; the high uniformity of the concentration of the obtained concentrated solution of the test body fluid; the high detection sensitivity and high S / N ratio in the test body fluid examination method of the present invention.

[0093] [1] Methods for concentrating the body fluids being tested

[0094] The method for concentrating the tested bodily fluid of the present invention (hereinafter also referred to as "the concentration method of the present invention") comprises, in sequence:

[0095] The test fluid injection process involves injecting the test fluid, which is an aqueous solution containing a high molecular weight polymer, into a cylinder containing particulate superabsorbent polymer.

[0096] In the water absorption process, the water contained in the test fluid injected into the above-mentioned cylinder is absorbed by the superabsorbent polymer contained in the above-mentioned cylinder, and a test fluid concentrate, which is a concentrate of the test fluid, is generated in the above-mentioned cylinder.

[0097] The liquid addition process (hereinafter also referred to as the "extract addition process") involves adding a smaller amount of liquid (hereinafter also referred to as the "extract") to the above-mentioned test body fluid concentrate than the test body fluid injected into the cylinder in the above-mentioned test body fluid injection process; and

[0098] In the removal process, the piston is inserted into the cylinder, thereby removing the concentrated body fluid of the test fluid, which is the concentrated body fluid of the test fluid, through the hole at the front end of the piston. The piston can be inserted into the cylinder and has a front end with a hole that is smaller than the particle size of the superabsorbent polymer after water absorption.

[0099] First, the concentration method of the present invention will be described with reference to the accompanying drawings.

[0100] Figure 1 Figures 1A to 1E ( ) is a schematic cross-sectional view showing one way of concentrating the method of the present invention in the order of the processes.

[0101] First, in the process of injecting the test fluid, the fluid is injected from the opening 216 into the cylinder 211 containing particulate superabsorbent polymer 230. Figure 1A 240 ml of the tested body fluid was injected into the sample. Figure 1B ).

[0102] Then, in the water absorption process, the water contained in the test fluid 240 is absorbed by the superabsorbent polymer 230, and a test fluid concentrate 246, which is a concentrate of the test fluid 240, is generated in the cylinder 211 (the superabsorbent polymer 230 becomes a swollen superabsorbent polymer 232). Figure 1C ).

[0103] Next, in the extract addition step, a smaller amount of extract 250 is added than the amount of the test body fluid injected into cylinder 211 in the test body fluid injection step. Figure 1D ).

[0104] Furthermore, during the extraction process, the piston 220 is inserted into the cylinder 211 through the opening 216, thereby extracting the concentrated body fluid 248, which is a concentrate of the body fluid 240, through the hole 222 at the front end 221 of the piston 220. The piston 220 is insertable into the cylinder 211 and has a front end 221 with a hole 222 smaller than the particle size of the superabsorbent polymer 230 after water absorption (the particle size of the swollen superabsorbent polymer 232). Figure 1E ).

[0105] The following is a description of each process.

[0106] [Subject fluid injection procedure]

[0107] As described above, the test fluid injection process is a process of injecting the test fluid, which is an aqueous solution containing a polymer, into a cylinder containing particulate superabsorbent polymer.

[0108] [Cylinder Block]

[0109] The shape of the cylinder is not particularly limited, but a cylindrical shape is preferred.

[0110] Typically, one end of the cylinder block along its length is closed (bottom surface), while the other end is open (opening).

[0111] There are no particular restrictions on the material of the cylinder body, but thermoplastic resins are preferred from the perspective of being able to be injection molded, inexpensive, and mass-produced. Specifically, from the perspective of having a certain degree of hardness, polypropylene, acrylic acid, polyacetal, polyamide, polyethylene, polyethylene terephthalate, polycarbonate, polystyrene, polyphenylene sulfide, polybutene terephthalate, polyvinyl chloride, ABS resin (acrylonitrile-butadiene-styrene copolymer), and AS resin (acrylonitrile-styrene copolymer) are preferred.

[0112] [Particulate superabsorbent polymer]

[0113] There are no particular limitations on the particulate superabsorbent polymer (SAP) contained in the above-mentioned cylinder, but from the perspective of superior effects of the present invention, polymers based on polyacrylic acid, polyacrylamide, cellulose or polyethylene oxide are preferred.

[0114] <Swelling ratio>

[0115] There is no particular limitation on the swelling ratio of the above-mentioned superabsorbent polymer. However, considering the superior effects of the present invention, it is preferable to be greater than 0.2 g / g and less than 800 g / g, more preferably 1.0 g / g or more and less than 600 g / g, even more preferably 10 g / g or more and less than 500 g / g, and especially preferably 20 g / g or more and less than 100 g / g.

[0116] Here, the swelling ratio is defined as the value of "the mass (g) of water held by 1g of superabsorbent polymer".

[0117] (Method for determining the swelling ratio)

[0118] The mass of a superabsorbent polymer (SAP) stored at 25°C and 5% RH (relative humidity) for 10 days was determined, and then it was immediately immersed in a large amount of distilled water. After 120 minutes, the SAP was removed, the surface water was removed, and the mass was determined again. The swelling ratio was calculated using the following formula.

[0119] {(mass after water absorption (g) - initial mass before water absorption (g)) / initial mass before water absorption (g)}

[0120] There are no particular limitations on the methods for adjusting the swelling ratio to the specific range mentioned above. Examples include changing the type of polymer, changing the molecular weight of the polymer, changing the degree of crosslinking, and changing the particle size.

[0121] <Water absorption rate>

[0122] There is no particular limitation on the water absorption rate of the superabsorbent polymer, but for reasons of superior effect of the present invention, it is preferable to be 0.01 g / min or more and 40 g / min or less per 1 g of superabsorbent polymer, and more preferably 0.02 g / min or more and 40 g / min or less per 1 g of superabsorbent polymer.

[0123] The water absorption rate was measured as follows.

[0124] The mass (weight M0, unit g) of a superabsorbent polymer stored at 25℃ and 5% RH (relative humidity) for 10 days was determined, and then it was immediately immersed in a large amount of distilled water. After 10 minutes, the superabsorbent polymer was removed, the surface water was removed, and the mass (mass M) was determined. 10 After the mass was measured, it was immediately immersed again in a large amount of distilled water. After 10 minutes, the superabsorbent polymer was removed, the surface water was removed, and the mass was measured again (mass M). 20 ). Determining mass M 20 Then, immediately immerse it again in a large amount of distilled water. After 10 minutes, remove the superabsorbent polymer, remove the surface water, and measure the weight again (mass M). 30 ).

[0125] Water absorption is defined as follows.

[0126] Water absorption in 10 minutes: ΔM10=(M 10 Water absorption in minutes: ΔM20 = (M0) / M020 20 Water absorption capacity in 30 minutes: ΔM30=(M) / M0 30 -M0) / M0

[0127] Using the water absorption rate defined above, the water absorption rate is calculated as follows.

[0128] Plot three points on the XY plane as the horizontal axis for time (x = 10, 20, 30; in minutes) and the vertical axis for water absorption (y = ΔM10, ΔM20, ΔM30; in g water / g polymer). The slope of the linear approximation of water absorption relative to time using the least squares method is taken as the water absorption rate per unit time (minutes).

[0129] <Particle size>

[0130] For reasons of superior effects of the present invention, the particle size of the superabsorbent polymer is preferably 10 mm or less, more preferably 8 mm or less, and even more preferably 5 mm or less. For reasons of superior effects of the present invention, 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 1 mm or more.

[0131] Here, the aforementioned particle size can be obtained by measuring the diameter of 50 polymer particles using an optical microscope and calculating their arithmetic mean.

[0132] [A binding substance that specifically binds to high molecular weight substances contained in biological fluids]

[0133] For reasons of superior effects, the aforementioned cylinder preferably further contains a binding substance that specifically binds to the high molecular weight contained in the biological fluids of the test fluid, as described later. When the cylinder contains this binding substance, for example, during the simultaneous concentration of the test fluid and the antigen-antibody reaction, a complex of antigen and labeled antibody in the test fluid is formed in a concentrated state, thereby improving detection sensitivity.

[0134] Examples of the aforementioned binding substances include, for instance, the first binding substance described later (especially an antibody). That is, in this invention, for reasons of superiority such as the effects of the invention, it is preferable that the polymer contained in the aforementioned biological fluid is an antigen, and the aforementioned binding substance is an antibody.

[0135] From the perspective of superior effects of the present invention, the above-mentioned combined substance is preferably included in the cylinder as a composite with the marking substance.

[0136] Examples of such complexes include labeled antibodies.

[0137] Here, labeled antibody refers to an antibody that binds to a detectable labeled substance. The labeled substance is, for example, a detectable substance that can be directly detected, such as 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, and is a substance or state containing enzymes that form a luminescent or chromogenic body by interacting with a luminescent or chromogenic precursor.

[0138] For reasons of superior effect of the present invention, the labeled antibody is preferably an antibody modified with metal particles that exhibit a bright hue when irradiated with electromagnetic waves such as visible light. For reasons of superior effect of the present invention, the metal particles are more preferably gold particles. For reasons of superior effect of the present invention, the labeled antibody is preferably an antibody labeled with gold particles, that is, gold particles modified with antibodies (modified gold particles described later).

[0139] The aforementioned labeled antibody can also be included in the aforementioned cylinder as a pad (gold colloidal holding pad) holding modified gold colloidal particles that are modified with the antibody.

[0140] [Casein, Tris(hydroxymethyl)methylglycine]

[0141] From the perspective of superior effects of the present invention, the cylinder body preferably further contains at least one of the group consisting of casein and tris(hydroxymethyl)glycine, and more preferably contains both casein and tris(hydroxymethyl)glycine.

[0142] Casein is believed to have the effect of inhibiting false positives. Furthermore, false positives are more likely to occur when the pH of the tested body fluids, such as urine, is acidic; however, tris(hydroxymethyl)glycine is believed to have the effect of adjusting the pH to neutral to alkaline, thus inhibiting false positives.

[0143] [Extract holding section]

[0144] For reasons of superior effects of the present invention, as shown in the preferred embodiment described later, the cylinder preferably has a liquid holding portion (hereinafter also referred to as "extract holding portion") at the bottom for holding the extract described later. The extract holding portion will be described later.

[0145] [The body fluid being tested]

[0146] The tested bodily fluid is an aqueous solution containing high molecular weight molecules. Preferably, it is an aqueous solution containing high molecular weight molecules found in biological bodily fluids.

[0147] Specific examples of the tested bodily fluids include animal (especially human) bodily fluids (e.g., blood, serum, plasma, cerebrospinal fluid, tears, sweat, urine, pus, nasal discharge, or sputum), mouthwash, etc. Among these, serum, plasma, urine, and nasal discharge are preferred as the test subject containing antigens as high molecular weight molecules, and urine is particularly preferred for reasons of superiority such as the effects of the present invention.

[0148] <High molecules contained in biological fluids>

[0149] High molecules (especially antigens) contained in the aforementioned bodily fluids, primarily those useful for disease diagnosis, include bacteria, bacteria (e.g., Mycobacterium tuberculosis, and the lipoarabinomannan (LAM) contained in Mycobacterium tuberculosis), bacteria (bacteria), viruses (e.g., influenza virus), or their nucleoproteins, which are detected in bodily fluids. Furthermore, LAM is a major antigen in tuberculosis and is a major component of the cell membrane and cell wall, namely glycolipids.

[0150] From the perspective of superior effects of the present invention, the polymer contained in the above-mentioned biological fluid is preferably an antigen, more preferably a virus (especially an influenza virus) or LAM, and even more preferably LAM.

[0151] For reasons of superior effects and other considerations, the molecular weight of the polymer contained in the biological fluid is preferably 1000 or more, more preferably 2000 or more. Molecular weight is useful for assessing diseases in polymers, and when the structural formula of the polymer is known, theoretical values ​​calculated from the structural formula can be used. Furthermore, when the structural formula is not determined, it can be calculated using methods such as electrophoresis and comparison with substances of known molecular weight, or by liquid chromatography-mass spectrometry (LC-MS).

[0152] <Pretreatment of the body fluids being tested>

[0153] The aforementioned body fluid can be used directly, or in liquid form obtained by extracting antigens using an appropriate extraction solvent, and then in diluted form obtained by diluting the extracted liquid with an appropriate diluent, or in concentrated form obtained by a suitable method.

[0154] As the solvent for the above extraction, solvents commonly used in immunological analysis methods (e.g., water, physiological saline, or buffer solutions) or water-mixable organic solvents that can be directly used to carry out antigen-antibody reactions by dilution with the solvent can also be used.

[0155] [The ratio of superabsorbent polymer to the tested body fluid]

[0156] There is no particular limitation on the ratio of the superabsorbent polymer to the tested body fluid, but considering the superior effects of the present invention, the ratio is preferably 0.01 to 100 g, more preferably 0.1 to 50 g, relative to 1 mL of the tested body fluid.

[0157] [Water Absorption Process]

[0158] As described above, the water absorption process is a process in which the water contained in the test body fluid injected into the cylinder during the test body fluid injection process is absorbed by the superabsorbent polymer contained in the cylinder, and a test body fluid concentrate is generated in the cylinder as a concentrate of the test body fluid.

[0159] During the water absorption process, the water in the body fluid being tested is usually almost completely absorbed by the superabsorbent polymer.

[0160] [Concentrated body fluid sample]

[0161] As described above, in the water absorption process, a concentrated body fluid is generated in the cylinder as a concentrate of the tested body fluid.

[0162] When the test fluid and the superabsorbent polymer are mixed, the water in the test fluid is absorbed by the superabsorbent polymer. Conversely, the polymers (e.g., antigens) in the test fluid have a certain hydrodynamic radius, so the mesh structure on the surface of the superabsorbent polymer creates a sieve effect, making it difficult for them to be absorbed by the superabsorbent polymer. As a result, the polymers (e.g., antigens) in the test fluid are concentrated.

[0163] The concentrated body fluids being tested usually exist near the superabsorbent polymer in the form of high molecular weight precipitates or high-concentration solutions of high molecular weight polymers dissolved in trace amounts of residual liquid.

[0164] [Extract Addition Process]

[0165] As described above, the extract addition step is a step of adding a smaller amount of extract to the test body fluid concentrate generated in the above-mentioned water absorption step than the test body fluid injected into the cylinder in the above-mentioned test body fluid injection step.

[0166] The extract has the function of extracting (removing) the concentrated body fluid generated during the water absorption process.

[0167] There are no particular restrictions on the method of adding the extract; for example, methods such as adding it from the opening of the tank or using a dropper to deliver the extract to the bottom of the tank can be cited.

[0168] [Extract]

[0169] The extraction solution described above is not particularly limited, and its function can be acquired as needed by using buffers, surfactants, or other additives. For reasons of superior performance and other considerations, the extraction solution is preferably a buffer solution, more preferably PBS (Phosphate buffered salts).

[0170] Furthermore, as shown in the preferred manner described later, a portion of the body fluid being tested can also be used as the extraction solution.

[0171] From the viewpoint of concentrating the tested bodily fluid, the amount of extract is less than the amount of tested bodily fluid injected into the tank. Furthermore, as described later in the preferred embodiment, when a portion of the tested bodily fluid is used as the extract, the amount of extract will necessarily be less than the amount of tested bodily fluid injected into the tank.

[0172] The ratio of the amount of extract to the amount of body fluid injected into the cylinder (extract / body fluid) should be less than 100% by volume. However, for reasons of superiority such as the effect of the present invention, it is preferable to be 30% or less, more preferably 20% or less, and even more preferably 0.01% or more and 10% or less.

[0173] [Removal Process]

[0174] As described above, the extraction process involves inserting a piston into the cylinder after the water absorption process, thereby extracting the concentrated body fluid of the test fluid as a concentrated body fluid through the hole at the front end of the piston. The piston can be inserted into the cylinder and has a front end with a hole that is smaller than the particle size of the superabsorbent polymer after water absorption.

[0175] During the extraction process, the piston is inserted into the cylinder, and the perforated front end is pressed into the superabsorbent polymer. At this time, as the extract moves uniformly within the gaps in the superabsorbent polymer and gathers upwards, a uniform concentrate of the tested body fluid can be obtained through the stirring effect.

[0176] 〔piston〕

[0177] The piston described above can be inserted into the cylinder and has a front end with a hole that is smaller than the particle size of the superabsorbent polymer after water absorption.

[0178] There are no particular restrictions on the material of the piston, and its preferred method is the same as that of the cylinder.

[0179] As described above, the front end of the piston has pores smaller than the particle size of the superabsorbent polymer after water absorption. Furthermore, the particle size of the superabsorbent polymer after water absorption can be determined by measuring the diameter of 50 polymer particles and calculating their arithmetic mean.

[0180] From the perspective of superior effects of the present invention, the diameter of the hole at the aforementioned front end is preferably less than 1 / 2 of the particle size of the superabsorbent polymer after water absorption, more preferably less than 1 / 5, and even more preferably less than 1 / 10.

[0181] From the perspective of superior effects of the present invention, the diameter of the pore at the aforementioned front end is preferably smaller than the particle size of the superabsorbent polymer before water absorption.

[0182] From the perspective of superior effects of the present invention, the diameter of the hole at the front end is preferably 0.01 to 5 mm, more preferably 0.1 to 2 mm.

[0183] The number of holes at the aforementioned front end is not particularly limited, but is preferably 10 to 100, more preferably 20 to 50.

[0184] From the perspective of superior effects of the present invention, the ratio of the total area of ​​the holes in the front end portion to the area of ​​the front end portion is preferably 5% or more, more preferably 10% or more, and even more preferably 15% or more.

[0185] [Concentrated body fluid sample]

[0186] As described above, a concentrated solution of the tested body fluid can be obtained during the extraction process. The amount of the concentrated solution is approximately the same as the amount of the extract. That is, the concentration ratio of the concentrated solution is approximately the ratio of the tested body fluid to the extract. Therefore, by keeping the amounts of the tested body fluid and the extract constant, a concentrated solution of the tested body fluid with the desired concentration ratio can be obtained.

[0187] [A cover with a recycling port]

[0188] In the above-described extraction process, for reasons of superiority such as the effects of the present invention, it is preferable to further use a cap having a recovery port for recovering the concentrated body fluid extracted as described above.

[0189] As a specific example of the aforementioned cover, the cover used in Embodiment 1 described later can be cited as an example. Figure 9 ).

[0190] [Preferred method]

[0191] As a preferred embodiment of the concentration method of the present invention, the following embodiment can be cited: In the concentration method of the present invention described above,

[0192] The above-mentioned test fluid injection process involves injecting the test fluid into the cylinder while simultaneously retaining a portion of the injected test fluid in the cylinder as the extraction solution.

[0193] The water absorption process described above involves the absorption of water contained in the test fluid (excluding the test fluid held as the extractant) injected into the cylinder by a highly absorbent polymer contained in the cylinder, thereby generating a concentrate of the test fluid within the cylinder.

[0194] The above-mentioned extract addition process is a process of obtaining the above-mentioned body fluid concentrate by adding the body fluid held as the extract to the above-mentioned body fluid concentrate.

[0195] In the preferred method described above, since a portion of the body fluid being tested is used as the extraction solution, there is no need to prepare the extraction solution separately, which improves usability.

[0196] Furthermore, as specific examples of the preferred method described above, the following two methods (preferred method A and preferred method B) can be cited.

[0197] (1) Preferred method A

[0198] Preferred method A is as follows: In the above preferred method,

[0199] The aforementioned cylinder has an extraction liquid holding section at the bottom for holding the extraction liquid, and the aforementioned superabsorbent polymer is accommodated in the cylinder above and in contact with the extraction liquid holding section.

[0200] The above-mentioned test fluid injection process is a process in which the test fluid is injected into the cylinder, and a portion of the test fluid injected into the cylinder is retained as the extraction liquid in the extraction liquid holding section.

[0201] As a preferred method A, specific methods such as methods A1 to A2, which will be described later, can be cited as examples.

[0202] (2) Preferred method B (Method B)

[0203] Method B is as follows: In the above preferred method,

[0204] The aforementioned test fluid injection process involves simultaneously injecting the test fluid into the cylinder, inserting the piston into the cylinder, and fixing the piston at a position lower than the liquid level of the test fluid injected into the cylinder but higher than the superabsorbent polymer contained in the cylinder. This process retains the portion of the test fluid above the piston within the injected cylinder as the extraction solution within the cylinder.

[0205] The above-mentioned extract addition process involves pulling up the piston to introduce the test fluid above the piston through the hole in the piston to the area below the piston, thereby adding the test fluid above the piston to the test fluid concentrate.

[0206] [Method A1]

[0207] Method A1 is as follows: In the preferred method A above,

[0208] The extract holding section is the portion surrounded by the bottom of the cylinder and a partition wall. The partition wall is disposed on the inner circumferential surface of the cylinder in a manner that allows it to move along the length of the cylinder. The partition wall has pores smaller than the particle size of the superabsorbent polymer before water absorption.

[0209] The above-mentioned extract addition process involves moving the partition wall to the bottom of the cylinder, introducing the test fluid held in the extract holding section through the hole of the partition wall to the top of the partition wall, thereby adding the test fluid held in the extract holding section to the test fluid concentrate.

[0210] Method A1 is illustrated using the accompanying drawings.

[0211] Figure 2 Figures 2A-2E) is a schematic cross-sectional view of one method of representing the process sequence A1.

[0212] First, in the process of injecting the test fluid, the fluid is injected from the opening 216 into the cylinder 212 containing particulate superabsorbent polymer 230. Figure 2A 240 ml of the tested body fluid was injected into the sample. Figure 2B ).

[0213] Here, the cylinder body 212 has a partition wall 260, which is disposed on the inner circumferential surface of the cylinder body 212 in a manner that allows it to move along the length direction of the cylinder body 212. Furthermore, the partition wall 260 has pores 262 smaller than the particle size of the superabsorbent polymer 230 before water absorption. The superabsorbent polymer 230 is contained within the cylinder body 212 above and in contact with the partition wall 260.

[0214] As described above, when the test fluid 240 is injected into the cylinder 212, a portion of the test fluid 240 is introduced below the partition wall 260 through the hole 262. Furthermore, in the water absorption process described later, the water contained in the test fluid 242 above the partition wall 260 is absorbed by the superabsorbent polymer 230, while the water contained in the test fluid 241 below the partition wall 260 is not absorbed by the superabsorbent polymer 230.

[0215] That is, the test fluid injection process is a process in which, while injecting the test fluid 240 into the cylinder 212, a portion of the test fluid injected into the cylinder 212 (test fluid 241) is used as the extractant added in the extractant addition process described later, and is held in the part (extractant holding part) surrounded by the bottom 217 and the partition wall 260 of the cylinder 212.

[0216] Then, in the water absorption process, the water contained in the body fluid 242 (the body fluid 242 in the body fluid 240 that exists only above the partition 260, excluding the body fluid 241 held as the extractant) is absorbed by the superabsorbent polymer 230, and a body fluid concentrate 246 is generated in the cylinder 211 as a concentrate of the body fluid 242 (the superabsorbent polymer 230 becomes a swollen superabsorbent polymer 232). Figure 2C ).

[0217] Next, in the extraction liquid addition process, the partition wall 260 is moved to the bottom surface 218 of the cylinder 212, and the test liquid 241 held in the extraction liquid holding section is introduced through the hole 262 of the partition wall 260 to the top of the partition wall 260, thereby adding the test liquid 241 held in the extraction liquid holding section to the test liquid concentrate 246. Figure 2D Additionally, in Figure 2D In this process, the piston 220, used in the extraction process described later, is inserted into the cylinder 212 through the opening 216, thereby pressing the superabsorbent polymer 232 downward and moving the partition wall 260 to the bottom surface 218 of the cylinder 212. However, the method of moving the partition wall 260 is not limited to this; for example, a mechanism for moving the partition wall 260 can be provided independently of the piston 220. Furthermore, instead of moving the partition wall 260, the bottom surface 218 can be moved along the length of the cylinder 212, moving the bottom surface 218 to the partition wall 260. The test fluid 241 held in the extraction liquid holding section is introduced through the hole 262 of the partition wall 260 to the top of the partition wall 260, thereby adding the test fluid 241 held in the extraction liquid holding section to the test fluid concentrate 246.

[0218] Furthermore, during the extraction process, the piston 220 is inserted into the cylinder 212, thereby extracting the concentrated body fluid 248, which is a concentrated solution of the body fluid 240, through the hole 222 at the front end 221 of the piston 220. The piston 220 is insertable into the cylinder 212 and has a front end 221 with a hole 222 smaller than the particle size of the superabsorbent polymer 230 after water absorption (the particle size of the swollen superabsorbent polymer 232). Figure 2E ).

[0219] <Next door>

[0220] As described above, in method A1, the cylinder block has a partition wall.

[0221] There are no particular restrictions on the material of the aforementioned partition, and its preferred method is the same as that of the aforementioned cylinder.

[0222] As described above, the partition wall has pores smaller than the particle size of the superabsorbent polymer before it absorbs water. Therefore, the superabsorbent polymer will not fall below the partition wall.

[0223] From the perspective of superior effects of the present invention, the diameter of the pores in the aforementioned partition is preferably less than 2 / 3 of the particle size of the superabsorbent polymer before water absorption.

[0224] From the perspective of superior effects of the present invention, the diameter of the hole in the partition wall is preferably 0.05 to 5 mm, more preferably 0.1 to 3 mm, and even more preferably 0.2 to 2 mm.

[0225] The number of holes in the partition wall at the front end is not particularly limited, but is preferably 10 to 100, more preferably 20 to 50.

[0226] From the perspective of superior effects of the present invention, the ratio of the total area of ​​the holes in the partition wall to the area of ​​the partition wall is preferably 5% or more, more preferably 10% or more, and even more preferably 15% or more.

[0227] [Method A2]

[0228] Method A2 is as follows: In the preferred method A above,

[0229] The extract holding section is formed by the pores of a porous resin housed at the bottom of the cylinder. The pores of the resin are smaller than the particle size of the superabsorbent polymer before water absorption.

[0230] The above-mentioned extract addition process involves crushing the resin and introducing the test fluid held in the extract holding section through the pores of the resin onto the resin, thereby adding the test fluid held in the extract holding section to the test fluid concentrate.

[0231] Method A2 is illustrated using the accompanying drawings.

[0232] Figure 3 Figures 3A-3E ) is a schematic cross-sectional view of one method of representing the process sequence A2.

[0233] First, in the process of injecting the test fluid, the fluid is injected from the opening 216 into the cylinder 213 containing particulate superabsorbent polymer 230. Figure 3A Inject the test fluid into () Figure 3B ).

[0234] Here, a porous synthetic resin 270 is contained in the bottom 217 of the cylinder 213. The pores (not shown) of the synthetic resin 270 are smaller than the particle size of the superabsorbent polymer 230 before water absorption. Furthermore, the superabsorbent polymer 230 is contained in the cylinder 213 above and in contact with the synthetic resin 270.

[0235] As described above, when the test fluid is injected into the cylinder 213, a portion of the test fluid is introduced into the pores of the synthetic resin 270 (the synthetic resin 270 becomes the test fluid 241 (not shown) which is introduced into the pores as part of the test fluid 241). Furthermore, in the water absorption process described later, the water contained in the test fluid 242, which exists only above the synthetic resin 270, is absorbed by the superabsorbent polymer 230, while the water contained in the test fluid 241, which exists within the pores of the synthetic resin 270, is not absorbed by the superabsorbent polymer 230.

[0236] That is, the test fluid injection process is a process in which, while injecting the test fluid into the cylinder 213, a portion of the test fluid injected into the cylinder 213 (test fluid 241) is used as an extractant added in the extractant addition process described later, and is held in the pores (extractant holding part) of the porous synthetic resin 270 contained in the bottom 217 of the cylinder 213.

[0237] Then, in the water absorption process, the water contained in the test body fluid 242 (the test body fluid 242 other than the test body fluid 241 held as the extractant) which exists only above the synthetic resin 270 is absorbed by the superabsorbent polymer 230, and a test body fluid concentrate 246, which is a concentrate of the test body fluid 242, is generated in the cylinder 213 (the superabsorbent polymer 230 becomes a swollen superabsorbent polymer 232). Figure 3C ).

[0238] Next, in the extract addition step, the synthetic resin 270 is crushed, and the test fluid 241 held in the extract holding section is introduced through the holes of the synthetic resin 270 to the top of the synthetic resin 270, thereby adding the test fluid 241 held in the extract holding section to the test fluid concentrate 246. Figure 3D ).

[0239] In addition, Figure 3D In this process, the piston 220 used in the removal process described later is inserted into the cylinder 213 through the opening 216, thereby pressing the superabsorbent polymer 232 downward and crushing the synthetic resin 270. However, the method of crushing the synthetic resin 270 is not limited to this. For example, a mechanism for crushing the synthetic resin 270 may be provided independently of the piston 220.

[0240] Furthermore, during the retrieval process, the piston 220 is inserted into the cylinder 213, thereby retrieving the concentrated body fluid 248, which is a concentrated solution of the tested body fluid, through the hole 222 at the front end 221 of the piston 220. The piston 220 is insertable into the cylinder 213 and has a front end 221 with a hole 222 smaller than the particle size of the superabsorbent polymer 230 after water absorption (the particle size of the swollen superabsorbent polymer 232). Figure 3E ).

[0241] <Resin>

[0242] As described above, in method A2, the cylinder body is made of porous resin.

[0243] As the aforementioned resin, any resin that exists in nature or a synthetic resin can be used, but from the viewpoint of ease of molding or the ability to produce in large quantities and at low cost, a synthetic resin is preferred.

[0244] There are no particular restrictions on the material of the aforementioned synthetic resins; polyvinyl alcohol (PVA) can be cited as a specific example.

[0245] From the perspective of superior effects of the present invention, the above-mentioned resin is preferably a sponge.

[0246] The pores of the resin are smaller than the particle size of the superabsorbent polymer before it absorbs water. Therefore, the superabsorbent polymer will not enter the resin.

[0247] The porosity (volume of voids / volume of resin containing voids) of the above-mentioned resin is not particularly limited, but from the perspective of superior effects of the present invention, it is preferably 50% or more, more preferably 70% or more and 99% or less, and even more preferably 70% or more and 98% or less.

[0248] [Method B]

[0249] Method B is illustrated using the accompanying drawings.

[0250] Figure 4 Figures 4A-4E () is a schematic cross-sectional view of method B, which represents the process sequence.

[0251] First, in the process of injecting the test fluid, the fluid is injected from the opening 216 into the cylinder 214 containing particulate superabsorbent polymer 230. Figure 4A While injecting the test fluid 240 into the cylinder, the piston 224 is inserted into the cylinder 214, and the front end 221 of the piston 224 is fixed at a position lower than the liquid level 244 of the test fluid 240 injected into the cylinder 214 and higher than the superabsorbent polymer 230 contained in the cylinder 214 (hereinafter also referred to as "position A"). The piston 224 can be inserted into the cylinder 214 and has a front end 221 with orifices 222 smaller than the particle size of the superabsorbent polymer 230 after water absorption. Figure 4B Additionally, the cylinder 214 and piston 224 are equipped with a piston position fixing mechanism (not shown) that overcomes the pressure of water absorption and expansion associated with the highly absorbent polymer 230 and fixes the front end 221 of the piston 224 at position A.

[0252] As described above, when the test fluid 240 is injected into the cylinder 214, the piston 224 is inserted into the cylinder 214, and the front end 221 of the piston 224 is fixed at position A, a portion of the test fluid is introduced through the hole 222 of the front end 221 of the piston 224 to the area above the front end 221 of the piston 224. Furthermore, in the water absorption process described later, the water contained in the test fluid 242 present only below the front end 221 of the piston 224 in the test fluid 240 is absorbed by the superabsorbent polymer 230, while the water contained in the test fluid 241 present above the front end 221 of the piston 224 in the test fluid 240 is not absorbed by the superabsorbent polymer.

[0253] That is, the test fluid injection process involves inserting the piston 224 into the cylinder 214 and fixing the front end 221 of the piston 224 at position A, thereby retaining the test fluid 241 present above the front end 221 of the piston 224 in the test fluid 240 injected into the cylinder 214 as the extractant added in the extractant addition process described later.

[0254] Then, in the water absorption process, the water contained in the body fluid 242 (the body fluid 242 in the body fluid 240 that exists only below the front end 221 of the piston 224, excluding the body fluid 241 held as the extractant) in the body fluid 240 is absorbed by the superabsorbent polymer 230, and a body fluid concentrate 246, which is a concentrate of the body fluid 242, is generated in the cylinder 214 (the superabsorbent polymer 230 becomes a swollen superabsorbent polymer 232). Figure 4C ).

[0255] Next, in the extraction liquid addition process, the piston 224 is pulled up, and the test fluid 241 existing above the front end 221 of the piston 224 is introduced through the hole 222 of the front end 221 of the piston 224 to below the front end 221 of the piston 224, thereby adding the test fluid 241 existing above the front end 221 of the piston 224 to the test fluid concentrate 246. Figure 4D ).

[0256] Furthermore, during the retrieval process, piston 224 is inserted into cylinder 214, thereby retrieving the concentrated body fluid 248, which is the concentrated body fluid 240, through the hole 222 at the front end 221 of piston 224. Figure 4E ).

[0257] <Piston Position Fixing Mechanism>

[0258] As described above, in the above-described method B, the cylinder and the piston are provided with a piston position fixing mechanism, which overcomes the pressure accompanying the water absorption and expansion of the superabsorbent polymer and fixes the front end of the piston at the above-described position A.

[0259] The piston position fixing mechanism described above is not particularly limited; for example, Embodiment 4 described below can be cited. Figure 10 The mechanism shown is as follows: the cylinder has a notch, the piston has a protrusion, the piston is inserted into the cylinder, and the protrusion of the piston is hung on the notch of the cylinder, thereby overcoming the pressure of water absorption and expansion associated with the superabsorbent polymer, and fixing the front end of the piston at position A.

[0260] [2] Concentration device

[0261] Next, the concentration device (hereinafter also referred to as "the concentration device of the present invention") used in the concentration method of the present invention will be described.

[0262] The concentrating device of the present invention comprises a cylinder containing particulate superabsorbent polymer and a piston that can be inserted into the cylinder, for concentrating the test fluid, which is an aqueous solution containing the polymer.

[0263] The piston described above has pores smaller than the particle size of the superabsorbent polymer after water absorption.

[0264] First, the condensation device of the present invention will be described using the accompanying drawings.

[0265] Figure 5 This is a schematic cross-sectional view of one embodiment of the condensation device of the present invention.

[0266] like Figure 5 As shown, the concentrator 201 includes a cylinder 211 containing a superabsorbent polymer 230 and a piston 220 that can be inserted into the cylinder 211. The piston 220 has a front end portion 221 having orifices 222 smaller than the particle size of the superabsorbent polymer 230 after water absorption.

[0267] [Preferred method]

[0268] As specific embodiments of the condensation device of the present invention, the following two embodiments (preferred embodiment A and preferred embodiment B) can be cited as examples.

[0269] (1) Preferred method A

[0270] Preferred method A is a concentration device comprising a cylinder containing particulate superabsorbent polymer and a piston that can be inserted into the cylinder, for concentrating the test fluid, which is an aqueous solution containing the polymer.

[0271] The aforementioned cylinder has an extraction liquid holding section at its bottom for holding a portion of the test fluid injected into the cylinder as an extraction liquid. The volume of the extraction liquid holding section is smaller than the volume of the test fluid injected into the cylinder, excluding the test fluid held in the extraction liquid holding section.

[0272] The aforementioned superabsorbent polymer is contained in the cylinder above and in contact with the extract holding portion.

[0273] The piston described above has pores smaller than the particle size of the superabsorbent polymer after water absorption.

[0274] The aforementioned superabsorbent polymer absorbs the water contained in the test body fluid injected into the aforementioned cylinder, excluding the test body fluid held in the aforementioned extract holding section, and generates a test body fluid concentrate in the aforementioned cylinder as a concentrate of the test body fluid.

[0275] By adding the test body fluid held in the extraction solution holding section to the test body fluid concentrate, a test body fluid concentrate is generated as a concentrate of the test body fluid.

[0276] The test fluid concentrate is removed from the piston by inserting it into the cylinder.

[0277] As a specific example of the preferred method A described above, methods A1 to A2, which will be described later, can be cited.

[0278] (2) Preferred method B (Method B)

[0279] Method B is a concentration device comprising a cylinder containing particulate superabsorbent polymer and a piston that can be inserted into the cylinder, used to concentrate the test fluid, which is an aqueous solution containing the polymer.

[0280] The piston described above has a front end portion with pores smaller than the particle size of the superabsorbent polymer after water absorption.

[0281] The aforementioned cylinder has an extractant holding section, which holds a portion of the sample fluid injected into the upper part of the piston as extractant by fixing the front end of the piston at a position lower than the liquid level of the sample fluid injected into the cylinder and higher than the superabsorbent polymer contained in the cylinder.

[0282] The volume of the extraction liquid holding section is smaller than the volume of the test body fluid injected into the cylinder, excluding the test body fluid held in the extraction liquid holding section. The superabsorbent polymer is contained in the cylinder below the extraction liquid holding section via the piston.

[0283] The cylinder and piston described above are equipped with a piston position fixing mechanism, which overcomes the pressure caused by the water absorption and expansion of the superabsorbent polymer, and fixes the front end of the piston in the aforementioned position.

[0284] The aforementioned superabsorbent polymer absorbs the water contained in the test body fluid injected into the aforementioned cylinder, excluding the test body fluid held in the aforementioned extract holding section, and generates a test body fluid concentrate in the aforementioned cylinder as a concentrate of the test body fluid.

[0285] Release the piston position fixing mechanism and lift the piston. The extract above the front end of the piston is introduced through the hole at the front end of the piston to below the front end of the piston, thereby adding the body fluid above the piston to the concentrated body fluid sample.

[0286] By pulling the piston down into the cylinder again, the concentrated body fluid of the test fluid, which is the concentrated body fluid of the test fluid, is taken out from the hole at the front end of the piston.

[0287] [Method A1]

[0288] Method A1 is as follows: In the preferred method A above,

[0289] The extract holding section is the portion surrounded by the bottom of the cylinder and a partition wall. The partition wall is disposed on the inner circumferential surface of the cylinder in a manner that allows it to move along the length of the cylinder. The partition wall has pores smaller than the particle size of the superabsorbent polymer before water absorption.

[0290] By moving the aforementioned partition to the bottom surface of the cylinder, the sample fluid held in the extraction fluid holding section is introduced through the holes of the partition to the area above the partition.

[0291] The sample fluid introduced above the aforementioned partition is added to the aforementioned sample fluid concentrate.

[0292] Method A1 is illustrated using the accompanying drawings.

[0293] Figure 6 This is a schematic cross-sectional view of method A1.

[0294] like Figure 6 As shown, the concentrator 202 includes a cylinder 212 containing a superabsorbent polymer 230 and a piston 220 that can be inserted into the cylinder 212. The piston 220 has a front end portion 221 having orifices 222 smaller than the particle size of the superabsorbent polymer 230 after water absorption.

[0295] Here, the cylinder body 212 has a partition wall 260, which is disposed on the inner circumferential surface of the cylinder body 212 in a manner that allows it to move along the length direction of the cylinder body 212. Furthermore, the partition wall 260 has pores 262 smaller than the particle size of the superabsorbent polymer 230 before water absorption. The superabsorbent polymer 230 is contained within the cylinder body 212 above and in contact with the partition wall 260.

[0296] As described in mode A1 of the concentration method of the present invention, the portion surrounded by the bottom 217 and the partition wall 260 of the cylinder 212 becomes the extract holding section.

[0297] [Method A2]

[0298] Method A2 is as follows: In the preferred method A above,

[0299] The extract holding section is formed by the pores of a porous resin housed at the bottom of the cylinder. The pores of the resin are smaller than the particle size of the superabsorbent polymer before water absorption.

[0300] By crushing the resin, the sample fluid held in the extract holding section is introduced through the pores of the resin and directed above the resin.

[0301] The test fluid introduced onto the above resin is added to the above test fluid concentrate.

[0302] Method A2 is illustrated using the accompanying drawings.

[0303] Figure 7 This is a schematic cross-sectional view of method A2.

[0304] like Figure 7 As shown, the concentrator 203 includes a cylinder 213 containing a superabsorbent polymer 230 and a piston 220 that can be inserted into the cylinder 213. The piston 220 has a front end portion 221 having orifices 222 smaller than the particle size of the superabsorbent polymer 230 after water absorption.

[0305] Here, a porous synthetic resin 270 is contained at the bottom 217 of the aforementioned cylinder 213. Furthermore, the pores (not shown) of the synthetic resin 270 are smaller than the particle size of the superabsorbent polymer 230 before water absorption. The superabsorbent polymer 230 is contained in the cylinder 213 above and in contact with the synthetic resin 270.

[0306] As described in mode A2 of the concentration method of the present invention, the pores of the synthetic resin 270 serve as extract holding portions.

[0307] [Method B]

[0308] Method B is illustrated using the accompanying drawings.

[0309] Figure 8 This is a schematic cross-sectional view of method B.

[0310] like Figure 8 As shown, the concentrator 204 includes a cylinder 214 containing a superabsorbent polymer 230 and a piston 224 that can be inserted into the cylinder 214. The piston 224 has a front end portion 221 with orifices 222 smaller than the particle size of the superabsorbent polymer 230 after water absorption. The cylinder 214 and the piston 224 are equipped with a piston position fixing mechanism (not shown) that overcomes the pressure of the superabsorbent polymer 230 expanding after water absorption and fixes the front end portion 221 of the piston 224 at position A.

[0311] [A cover with a recycling port]

[0312] From the perspective of superior effects and other considerations, the concentration device of the present invention preferably also includes a cap having a recovery port for recovering the above-mentioned concentrated body fluid solution.

[0313] As a specific example of the aforementioned cover, the cover used in Embodiment 1 described later can be cited as an example. Figure 9 ).

[0314] [3] The method for examining the body fluid of the present invention

[0315] The method for examining bodily fluids of the present invention (hereinafter also referred to as "the method for examining bodily fluids of the present invention") detects macromolecules in a bodily fluid containing an aqueous solution of macromolecules, comprising the following steps:

[0316] In the concentration step, the above-described concentrated body fluid of the present invention is obtained using the concentration method of the present invention (the concentration method of the present invention described above); and

[0317] The testing process involves detecting the high molecular weight molecules in the obtained concentrated body fluid solution.

[0318] In the inspection method of the present invention, since the concentrated body fluid obtained by the concentration method of the present invention described above is used for the detection of high molecular weight, high detection sensitivity can be obtained.

[0319] [Concentration Process]

[0320] The method for obtaining the concentrated body fluid sample using the concentration method of the present invention is as described above.

[0321] [Inspection Process]

[0322] The testing process involves detecting high molecular weight molecules in the concentrated body fluid sample.

[0323] For reasons of superior performance and other considerations, the detection process is preferably performed using an antigen-antibody reaction method. Examples of such methods include enzyme immunoassay (EIA), solid-phase enzyme immunoassay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Western blotting, and immunochromatography. Among these, immunochromatography is preferred for reasons of superior performance and other considerations.

[0324] [Preferred method]

[0325] From the perspective of superior effects and other considerations, the inspection method of the present invention is preferably the following inspection method:

[0326] The body fluids tested were aqueous solutions that may contain antigens (high molecular weight).

[0327] The above-described concentration step involves using the concentration method of the present invention to concentrate an aqueous solution that may contain the above-described antigen to obtain an antigen concentrate (a concentrate of the body fluid being tested).

[0328] The above-mentioned detection procedure is a process of detecting the antigen in the above-mentioned antigen concentrate by using an immunochromatographic method of antigen-antibody reaction.

[0329] Therefore, considering the superior effects of the present invention, the above-mentioned detection process preferably includes:

[0330] The unfolding process involves unfolding a gold particle complex—a complex of the antigen in the aforementioned antigen concentrate and gold particles modified with a first binding substance capable of binding to the aforementioned antigen—on an insoluble carrier having a reaction site immobilized with a second binding substance capable of binding to the aforementioned antigen; and

[0331] The capture process involves capturing the gold particle complex at the reaction site of the insoluble carrier.

[0332] For reasons of superior effects of the present invention, the above detection process preferably further includes a silver amplification process, which amplifies the gold particle complex captured in the above capture process.

[0333] Here, considering the superior 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 more preferably both the first binding substance and the second binding substance are monoclonal antibodies.

[0334] Furthermore, the tested bodily fluids sometimes contain impurities such as salts. For example, in the case of urine, the tested bodily fluid may contain impurities such as urea, which are low-molecular-weight components. According to the researchers of this invention, when these impurities are concentrated along with the high-molecular-weight components present in bodily fluids, the antigen-antibody reaction is sometimes hindered, leading to a decrease in detection sensitivity. That is, it is known that the increased detection sensitivity achieved through concentration cannot always be fully realized.

[0335] Therefore, in the concentration device of the present invention used in the above-described concentration process, the swelling ratio of the superabsorbent polymer is preferably within the aforementioned preferred range, which allows for sufficient absorption of impurities. If it is within the aforementioned range, these impurities are absorbed by the superabsorbent polymer along with water, making it difficult for the detection sensitivity to decrease as described above to occur. As a result, it is believed that extremely high detection sensitivity can be achieved for polymers contained in biological fluids.

[0336] The steps of the preferred method described above (hereinafter also referred to as "the method of the present invention") will be described below.

[0337] [Development Process]

[0338] The unfolding process is a process in which a gold particle complex, which is a complex of an antigen obtained in the above-mentioned concentration process and gold particles modified with a first binding substance capable of binding with the above-mentioned antigen, is unfolded on an insoluble carrier having a reaction site with a second binding substance capable of binding with the above-mentioned antigen.

[0339] <Gold Particle Complex>

[0340] As described above, in the unfolding process, firstly, a gold particle complex is formed, which is a complex of the antigen in the antigen concentrate obtained in the above-described concentration process and gold particles modified with a first binding substance capable of binding to the antigen, i.e., modified gold particles. Alternatively, if a complex of the antigen and labeled antibody in the test body fluid is formed simultaneously with the concentration of the test body fluid, the antigen concentrate may be unfolded directly on an insoluble carrier.

[0341] (Modified gold particles)

[0342] Modified gold particles are gold particles modified with a first binding substance capable of binding to the aforementioned antigens.

[0343] (1)(gold particles)

[0344] There are no particular limitations on the gold particles, but gold colloidal particles are preferred for reasons such as the superior effects of the present invention.

[0345] In the case where the method of the present invention includes the silver amplification step described later, gold particles act as a catalyst for the reduction of silver ions in the silver amplification step.

[0346] From the perspective of superior effects of the present invention, the particle size of the gold particles is preferably 500 nm or less, more preferably 300 nm or less, even more preferably 200 nm or less, and especially preferably 100 nm or less.

[0347] There is no particular limitation on the lower limit of the particle size of the gold particles, but considering the superior effects of the present invention, it is preferred to be 1 nm or more, more preferably 2 nm or more, and even more preferably 5 nm or more.

[0348] Furthermore, particle size can be measured using commercially available particle size analyzers. Known methods for measuring particle size distribution include optical microscopy, confocal laser microscopy, electron microscopy, interatomic force microscopy, static light scattering, laser diffraction, dynamic light scattering, centrifugal sedimentation, electrical pulse measurement, chromatography, and ultrasonic attenuation. Apparatus corresponding to each principle is commercially available. Considering the particle size range and ease of measurement, dynamic light scattering is preferred as a method for measuring particle size. Examples of commercially available measuring apparatuses using dynamic light scattering include NANOTRAC UPA (Nikkiso Co., Ltd.), the LB-550 dynamic light scattering particle size distribution measuring apparatus (HORIBA, Ltd.), and the FPAR-1000 thick particle size analyzer (OTSUKA ELECTRONICS Co., LTD.). In this invention, the median particle size (d = 50) measured at a measurement temperature of 25°C is determined.

[0349] (2) First binding substance

[0350] The first binding substance is not particularly limited as long as it can bind to the antigens described above, but from the perspective of superior effects of the present invention, it is preferred to be a protein, more preferably an antibody (e.g., a polyclonal antibody or a monoclonal antibody), and from the viewpoint of achieving higher detection sensitivity, it is even more preferred to be a monoclonal antibody.

[0351] There are no particular limitations on the antibodies mentioned above. For example, antiserum prepared from the serum of an animal immunized with the antigen or immunoglobulin components purified from the antiserum can be used, and monoclonal antibodies or fragments thereof [e.g., F(ab')2, Fab, Fab', or Fv] obtained by cell fusion of spleen cells from an animal immunized with the antigen can be used. The preparation of these antibodies can be carried out by conventional methods.

[0352] As an example of the first binding substance when the antigen is the influenza virus, commercially available antibodies can be used, such as anti-influenza A monoclonal antibody (Anti-Influenza A SPTN-5 7307, Medix Biochemica) or anti-influenza A monoclonal antibody (manufactured by Bios Pacific Inc., clone number: A60010044P).

[0353] Furthermore, as an example of the first binding substance when the antigen is LAM, the A194-01 antibody described in International Publication No. 2017 / 139153 can be cited. The entire contents of International Publication No. 2017 / 139153 concerning the A194-01 antibody are incorporated herein by reference as part of the disclosure of this specification.

[0354] Another example of the first binding substance when the antigen is LAM is an antibody having the sequence MoAb1 described in paragraph

[0080] of International Publication No. 2013 / 129634. The entire contents of International Publication No. 2013 / 129634 concerning the MoAb1 antibody are incorporated herein by reference as part of this disclosure.

[0355] (3) Method for manufacturing modified gold particles

[0356] There are no particular limitations on the methods for manufacturing the modified gold particles described above, and well-known methods can be used. For example, one method is to introduce the SH group into the antibody by utilizing the chemical bonding between gold and the SH group, and then fix it by the Au-S bond generated on the Au surface when the SH bond breaks upon approaching the gold particle.

[0357] <Insoluble carrier>

[0358] The aforementioned insoluble carrier (porous carrier) is an insoluble carrier having a reaction site (test line) on which a second binding substance capable of binding to the aforementioned antigen is immobilized. The insoluble carrier can have multiple test lines depending on the type of antigen (e.g., test lines for influenza A virus and test lines for influenza B virus). Furthermore, to confirm the development of the aforementioned gold particle complex, the insoluble carrier may have a control line downstream of the test line. Moreover, in the case where a reducing agent solution is used in the silver amplification process described later, a chromogenic reagent immobilization line may be provided downstream of the test line to detect the reducing agent solution.

[0359] Specific examples of the aforementioned insoluble carriers include, for instance, those mentioned above. Figure 11The nitrocellulose membrane 300 shown has a gold colloid holding pad 301, a test line 302, a control line 303, and a colorimetric reagent immobilization line 304 from the upstream side. Here, the gold colloid holding pad 301 is a pad that holds gold particles modified with a first binding substance (modified gold particles), the test line 302 is a line with a second binding substance immobilized, the control line 303 is a line used to confirm the development, and the colorimetric reagent immobilization line 304 is a line used to detect the reducing agent solution described later. Here, "upstream side" and "downstream side" refer to the direction of development from the upstream side to the downstream side during the development of the gold particle complex.

[0360] As a more specific example of the aforementioned insoluble carrier (or immunochromatographic kit having the insoluble carrier), the insoluble carrier and immunochromatographic kit described in Japanese Patent No. 5728453 can be cited, and the entire contents of Japanese Patent No. 5728453 concerning the insoluble carrier and immunochromatographic kit are incorporated herein by reference as part of the disclosure of this specification.

[0361] (Insoluble carrier)

[0362] The insoluble carrier is preferably a porous carrier. In particular, for reasons of superior performance of the present invention, nitrocellulose membranes, cellulose membranes, acetylcellulose membranes, polysulfone membranes, polyethersulfone membranes, nylon membranes, glass fibers, nonwoven fabrics, cloths, or filaments are preferred, and nitrocellulose membranes are even more preferred.

[0363] (Second binding substance)

[0364] There are no particular restrictions on the second binding substance as long as it can bind to the aforementioned antigens.

[0365] Specific examples and preferred embodiments of the second binding substance may be given, for example, the same specific examples and preferred embodiments described in the first binding substance above. The second binding substance may be the same as or different from the first binding substance above, but from the perspective of superior effects of the present invention, a different substance is preferred.

[0366] Furthermore, when the first binding substance and the second binding substance are antibodies, from the perspective of superior effects of the present invention, it is preferable that the antibody as the first binding substance and the antibody as the second binding substance are different.

[0367] Furthermore, when both the first and second binding substances are antibodies, for reasons of superiority such as the effects of the present invention, it is preferable that the epitopes of the first binding substance (a portion of the antigen recognized by the first binding substance) and the epitopes of the second binding substance (a portion of the antigen recognized by the second binding substance) are different. The difference in antibody epitopes can be confirmed, for example, by ELISA (Enzyme-Linked ImmunoSurfact Assay).

[0368] <Expand>

[0369] There are no particular limitations on the method of developing the gold particle complex on an insoluble support with test lines; for example, preparations as described above can be used. Figure 11 The nitrocellulose membrane 300 shown (or an immunochromatographic kit having the nitrocellulose membrane 300) is used to drop the antigen concentrate obtained in the above concentration step onto the gold colloidal holding pad, as shown. Figure 11 The method shown utilizes the capillary phenomenon to move it from the upstream side to the downstream side, etc.

[0370] [Capture Process]

[0371] The capture process is a process of capturing the gold particle complex at the reaction site of the insoluble carrier.

[0372] As described above, since a second binding substance capable of binding to the antigen is fixed at the reaction site of the insoluble carrier, the gold particle complex (a complex of antigen and modified gold particles) that is developed on the insoluble carrier in the above-mentioned development process is captured at the reaction site (test line) of the insoluble carrier.

[0373] Because the captured gold particle complexes are colored by surface plasmons and other substances on the gold particles, they can be visually identified. Furthermore, the concentration of the captured complexes can be estimated using image analysis devices. This allows for the detection of antigens in the sample.

[0374] In addition, when the sample does not contain antigen, the aforementioned gold particle complex is not formed, so it is not captured at the reaction site of the insoluble carrier and is not stained.

[0375] [Silver Amplification Process]

[0376] The silver amplification process is a process of amplifying the gold particle complex captured in the above-mentioned capture process.

[0377] The silver amplification process involves imparting silver ions to the insoluble support following the capture process, thereby forming large silver particles within the gold particle complex captured at the reaction site of the insoluble support. More specifically, it involves using the gold particles of the aforementioned gold particle complex as a catalyst to reduce silver ions, forming silver particles (e.g., with a diameter of 10 μm or more).

[0378] As a result, the detection sensitivity of the captured gold particle complex is significantly improved.

[0379] Alternatively, the silver amplification process can be performed together with the development process, and the silver amplification process can also serve as the development process.

[0380] <Preferred Method>

[0381] There are no particular limitations on the method of imparting silver ions to the insoluble carrier after the above-mentioned capture process, but considering the superior effects of the present invention, the method of using the following reducing agent solution and the following silver amplification solution is preferred.

[0382] Furthermore, in addition to reducing agents and silver amplification solutions, cleaning solutions can also be used to clean complexes remaining in the insoluble carrier through specific binding reactions. The aforementioned reducing solutions can also be used as cleaning solutions.

[0383] (Reducing agent solution)

[0384] The aforementioned reducing agent solution contains a reducing agent capable of reducing silver ions. Any inorganic or organic material or mixture thereof can be used as the reducing agent, as long as it can reduce silver ions to silver. As an inorganic reducing agent, Fe is a preferred example. 2+ V 2+ and Ti 3+ Reducing metal salts and reducing metal complexes are metals whose oxidation states can be altered by metal ions. When using inorganic reducing agents, it is necessary to remove or neutralize the oxidized ions by complexing or reducing them. For example, when using Fe... 2+ In systems where citric acid or ethylenediaminetetraacetic acid (EDTA) is used as a reducing agent, Fe can be formed as an oxide. 3+ The complex is made harmless. In this invention, such an inorganic reducing agent is preferably used; as a more preferred embodiment of the invention, Fe is preferably used. 2+ Metal salts are used as reducing agents.

[0385] In addition, developing agents used in wet silver halide photographic materials (such as methyl gallate, hydroquinone, substituted hydroquinone, 3-pyrazolidineone, p-aminophenol, p-phenylenediamine, hindered phenol, methylamine oxime, azazine, catechol, pyrogallol, ascorbic acid (or its derivatives), and colorless dyes) and other materials that are obvious to those skilled in the art, such as those described in U.S. Patent No. 6,020,117, can be used as reducing agents.

[0386] As a reducing agent, ascorbic acid reducing agents are preferred. Useful ascorbic acid reducing agents contain ascorbic acid and its analogues, isomers and derivatives thereof, and preferably include, for example, D- or L-ascorbic acid and its sugar derivatives (e.g., γ-lactobionic ascorbic acid, gluconic acid ascorbic acid, alginate ascorbic acid, glucoheponic acid ascorbic acid, maltobionic ascorbic acid), sodium salts of ascorbic acid, potassium salts of ascorbic acid, isoascorbic acid (or L-erythromycin ascorbic acid) and its salts (e.g., alkali metal salts, ammonium salts or salts known in the art), enediol-type ascorbic acid, enamine-type ascorbic acid, thiol-enol-type ascorbic acid, etc., especially preferred are D, L or D,L-ascorbic acid (and its alkali metal salts) or isoascorbic acid (or its alkali metal salts), with sodium salts being preferred. Mixtures of these reducing agents can be used as needed.

[0387] From the perspective of superior effects of the present invention, the reducing agent liquid preferably flows in such a way that the angle between the spreading direction and the spreading direction of the reducing agent liquid in the spreading process is 0 degrees to 150 degrees, and more preferably flows in such a way that the angle between the spreading direction and the spreading direction of the reducing agent liquid in the spreading process is 0 degrees to 135 degrees.

[0388] In addition, as a method for adjusting the angle between the unfolding direction and the unfolding direction of the reducing agent liquid in the unfolding process, the method described in the embodiments of Japanese Patent Application Publication No. 2009-150869 can be cited as an example.

[0389] (Silver amplification solution)

[0390] The aforementioned silver amplification solution is a liquid containing a compound comprising silver ions. As the silver-containing compound, examples include organic silver salts, inorganic silver salts, or silver complexes. Preferably, silver nitrate, silver acetate, silver lactate, silver butyrate, and silver thiosulfate are silver-containing compounds with high solubility in solvents such as water. Silver nitrate is particularly preferred. As the silver complex, silver complexes coordinated to ligands having water-soluble groups such as hydroxyl and sulfone groups are preferred; examples include silver hydroxysulfide.

[0391] Organic silver salts, inorganic silver salts, or silver complexes are contained in the silver amplification solution at a concentration of 0.001 mol / L to 5 mol / L, preferably at a concentration of 0.005 mol / L to 3 mol / L, and more preferably at a concentration of 0.01 mol / L to 1 mol / L.

[0392] Examples of additives for silver amplification solutions include buffers, preservatives such as antioxidants or organic stabilizers, and rate modifiers. Buffers such as acetic acid, citric acid, sodium hydroxide, or their salts, or buffers using tris(hydroxymethyl)aminomethane, or other buffers commonly used in chemical experiments, can be used. Appropriate use of these buffers allows the solution to be adjusted to the optimal pH for its amplification. Furthermore, alkylamines, particularly dodecylamine, can be used as antifogging agents. Surfactants, particularly C9H, can be used to improve the solubility of these additives. 19 -C6H4-O-(CH2CH2O) 50 H.

[0393] For reasons of superior effects of the present invention, the silver amplification solution preferably flows in the opposite direction to the above-described development process, and more preferably flows in such a way that the angle between the development direction in the development process and the development direction of the reducing agent solution is 45 degrees to 180 degrees.

[0394] In addition, as a method for adjusting the angle between the development direction and the development direction of the silver amplification solution in the development process, the method described in the embodiment of Japanese Patent Application Publication No. 2009-150869 can be cited as an example.

[0395] [4] Test kit

[0396] The test kit of the present invention is used to detect macromolecules in a test body fluid that is an aqueous solution containing macromolecules, and comprises:

[0397] The above-described condensation device of the present invention; and

[0398] The detection device detects high molecules in the concentrated body fluid obtained by the concentration method of the present invention described above.

[0399] [Concentrated Devices]

[0400] The condensation device of the present invention is as described above.

[0401] [Detection Devices]

[0402] Considering the superior effects of the present invention, the detection device described above is preferably an immunochromatographic analyzer.

[0403] From the perspective of superior effects of the present invention, the detection device described above preferably comprises:

[0404] The test strip includes an insoluble carrier having a test region for detecting high molecular weights contained in biological fluids; a first canister and a second canister, respectively sealed with a first amplification solution and a second amplification solution for amplifying the test signal in the test region; and a casing containing the test strip, the first canister, and the second canister.

[0405] [Preferred method]

[0406] From the perspective of superior effects of the present invention, the above-described detection device is preferably an immunochromatographic reagent kit (hereinafter also referred to as "the immunochromatographic reagent kit of the present invention" or simply "immunochromatographic reagent kit"):

[0407] An immunochromatographic reagent kit for detecting a target substance (a macromolecule contained in biological body fluids) in a sample solution (body fluid being tested), comprising:

[0408] Test strips for testing contain an insoluble carrier that allows the sample solution to spread and has a test area containing the substance being tested;

[0409] The first and second containers are respectively sealed with a first amplification solution and a second amplification solution for amplifying detection signals in the inspection area, and each has a side with a sheet component; and

[0410] The outer casing contains test strips, canister 1, and canister 2.

[0411] The outer casing comprises the following components: a lower casing with a receiving portion for holding test strips; an upper casing joined to the lower casing at its periphery; and an intermediate component disposed between the upper and lower casings.

[0412] The intermediate component has a fracture portion that causes the sheet component of the first can to break, and the fracture portion faces the sheet component of the first can.

[0413] The upper shell is composed of the following components: a first convex deformable portion, which deforms the first can side by applying pressure from the outside to the portion opposite to the first can, and breaks the sheet component of the first can by using the fracture portion of the intermediate component; and a second convex deformable portion, which deforms towards the second can side by applying pressure from the outside to the portion opposite to the second can, and breaks the sheet component of the second can.

[0414] In the immunochromatographic kit of the present invention, preferably, the first convex deformable portion is moved to the position where the sheet component is broken by the fracture portion of the intermediate component by applying pressure.

[0415] At this time, it is preferable that the upper shell has two protrusions that stand upright toward the first can when pressure is applied to the first convex deformable part, and move it.

[0416] In the immunochromatographic kit of the present invention, the first convex deformed portion preferably has a centrally symmetrical mountain-shaped shape.

[0417] Furthermore, it is preferable that the two protrusions are arranged symmetrically with respect to the top of the mountain-shaped structure.

[0418] Furthermore, it is preferable that the two protrusions are formed independently on the sloping surface that sandwiches the top of the mountain-shaped structure.

[0419] In the immunochromatographic reagent kit of the present invention, when the first convex deformable portion has the above-mentioned two protrusions, it is preferable that the two protrusions are arranged symmetrically with respect to the center of the contact surface of the first container.

[0420] Furthermore, it is preferable that the two protrusions are positioned further to the end than half the distance from the center of the contact surface of the first can to the end.

[0421] In addition, in this specification, a convex deformable part refers to a convex shape when viewed from the outside of the immunochromatographic kit, and similarly, a mountain-shaped shape refers to a mountain-shaped shape when viewed from the outside.

[0422] In the immunochromatographic reagent kit of the present invention, when the first convex deformable portion has the above-mentioned two protrusions, it can be configured such that the front end of each of the two protrusions abuts against the first container, slowly displaces to the end side, and moves the first container.

[0423] In the immunochromatographic reagent kit of the present invention, the flexural modulus of the material constituting the first convex deformable part is preferably 50 MPa to 350 MPa.

[0424] Furthermore, it is preferred that the flexural modulus of the material constituting the upper shell is 50 MPa to 350 MPa, and the flexural modulus of the material constituting the lower shell is 500 MPa to 900 MPa.

[0425] In the immunochromatographic reagent kit of the present invention, the upper shell is preferably formed integrally by injection molding to form the first convex deformable portion and the second convex deformable portion.

[0426] In the immunochromatographic reagent kit of the present invention, the upper shell includes: a first convex deformable portion, which deforms the first canister side by applying pressure from the outside to the portion opposite to the first canister, and the sheet component of the first canister is broken by the fracture portion of the intermediate component; and a second convex deformable portion, which deforms towards the second canister side by applying pressure from the outside to the portion opposite to the second canister, and the sheet component of the second canister is broken. Furthermore, by applying pressure to the two convex deformable portions with a finger or the like, deformation is achieved, and the sheet component of the canister can be broken, allowing the amplification solution to be supplied to the test strip. Therefore, the amplification reaction can proceed normally even without a dedicated analytical device requiring a power source. Thus, the immunochromatographic reagent kit of the present invention is particularly useful in emergencies or disasters where a dedicated analytical device is unavailable or cannot be used.

[0427] Hereinafter, embodiments of the immunochromatographic reagent kit of the present invention will be described using the accompanying drawings, but the immunochromatographic reagent kit of the present invention is not limited thereto. Furthermore, for ease of visual identification, the scales and other details of the components in the drawings have been appropriately altered from the actual situation.

[0428] Figure 12 This is a schematic perspective view of the immunochromatographic reagent kit 100 according to an embodiment of the present invention. Figure 13 yes Figure 12 A schematic three-dimensional diagram of the breakdown of the immunochromatographic reagent kit 100.

[0429] like Figure 12 and Figure 13 As shown, the immunochromatographic reagent kit 100 of this embodiment comprises the following components within the outer casing 9: a test strip 1, including an insoluble carrier 2 (porous carrier 2) that spreads the sample solution and has a test area containing the analyte; and first canisters 40 and 45, which respectively contain first amplification solution 41 and second amplification solution 46 for amplifying the detection signal in the test area, and each canister has a side with a sheet component. The outer casing 9 comprises the following components: a lower casing 20, having a receiving portion 21 for holding the test strip 1; an upper casing 10, which is joined to the lower casing 20 at its periphery; and an intermediate component 30 disposed between the upper casing 10 and the lower casing 20. Furthermore, in describing the immunochromatographic reagent kit 100, the upper casing 10 side is defined as the upper side, and the lower casing 20 side is defined as the lower side.

[0430] The intermediate component 30 has a can housing 32 that houses the first can 40 and has an amplification solution filling hole on its bottom surface for dripping the first amplification solution 41 onto the insoluble carrier 2. Furthermore, a protruding fracture portion 34 is provided in the can housing 32 at the position of the sheet component 43 facing the first can 40, allowing the sheet component 43 to break. In this example, the first can 40 is positioned above the can housing 32 with the surface having its sheet component 43 as its lower surface, and the fracture portion 34 is provided on the bottom surface of the can housing 32 opposite to the sheet component 43 (see reference). Figure 14 ).

[0431] Furthermore, it includes a flow path forming section 35 extending downstream of the bottom surface of the can receiving section 32 of the intermediate component 30. The flow path forming section 35 is positioned above the inspection area L1, the confirmation area L2, and the amplification index area L3, and is formed of a transparent material so that these areas L1 to L3 can be visually identified.

[0432] The upper housing 10 has a first convex deformable portion 12, which deforms towards the first can 40 by applying pressure from the outside to the portion opposite to the first can 40, thereby causing the sheet component 43 of the first can 40 to break using the fracture portion 34 of the intermediate component 30. Furthermore, the upper housing 10 has a second convex deformable portion 14 on the portion opposite to the second can 45, which deforms towards the second can 45 by applying pressure from the outside, thereby causing the sheet component 48 of the second can 45 to break.

[0433] Furthermore, a sample liquid dispensing opening 16 is provided on the upper housing 10, through which the sample liquid is dispensed onto the mark holding pad 3 of the test strip 1. By adjusting the position of the mark holding pad 3 so that the opening 16 and the mark holding pad 3 are aligned, the sample liquid can be reliably dispensed onto the mark holding pad 3. Additionally, the upper housing 10 has an observation window 18 at a position corresponding to the flow path forming portion 35 of the intermediate component 30 for visually identifying three regions L1 to L3.

[0434] On the lower housing 20, there is an insoluble carrier container 21 for holding the insoluble carrier 2 and an absorbent pad container 22 for holding the absorbent pad 6 downstream of the insoluble carrier container 21. Furthermore, a second container container 24 for holding the second container 45 is provided upstream of the insoluble carrier container 21.

[0435] Figure 14 This is a schematic cross-sectional view showing the positional relationship between the test strip 1, the intermediate component 30, and the two containers 40 and 45. For example... Figure 14As shown, the test strip 1 includes: an insoluble carrier 2 for spreading the sample solution; a label holding pad 3 containing a labeling substance modified with a first substance capable of binding to the test substance fixed on the insoluble carrier 2; a delivery pad 4 for delivering a second amplification solution 46 disposed in contact with one end of the insoluble carrier 2 to the insoluble carrier 2; and an absorption pad 6 disposed in contact with the other end of the insoluble carrier 2. The insoluble carrier 2 is fixed and supported on a back adhesive sheet 7. Furthermore, the insoluble carrier 2, between the label holding pad 3 and the absorption pad 6, sequentially from the label holding pad 3 side, has a test area L1 containing the second substance that binds to the test substance, a confirmation area L2 containing a substance capable of binding to the first substance, and an amplification index area L3 containing a substance that reacts with the second amplification solution.

[0436] Additionally, in this specification, the insoluble carrier 2 forming the examination region L1, the confirmation region L2, and the amplification index region L3 is sometimes referred to as the chromatography carrier. Furthermore, in this specification, if... Figure 14 According to the description, the 4th side of the liquid delivery pad is defined as the upstream side, and the 6th side of the absorbent pad is defined as the downstream side.

[0437] The intermediate component 30 is located on the upper part of the downstream end of the test strip 1, and the first canister 40 is disposed in the canister receiving portion 32 of the intermediate component 30 with the sheet component 43 facing downward. The second canister 45 is disposed below the upstream end of the test strip 1 of the lower housing 20 with the sheet component 48 facing upward.

[0438] like Figure 14 As shown, a gap (void) D is formed between the back surface 36 of the flow path forming portion 35 of the intermediate component 30 and the insoluble carrier 2 of the test strip 1. This gap D is preferably in the range of 0.01 mm to 1 mm. If it is 0.01 mm or more, the amplification solution can be sufficiently impregnated; if it is 1 mm or less, capillary force can be utilized, allowing the first amplification solution 41 to uniformly fill the gap between the insoluble carrier 2 and the intermediate component 30.

[0439] A first can 40 containing the first amplification solution 41 is, for example, a container 42 made of resin material and having an opening on one side, which is covered and sealed by a breakable sheet component 43.

[0440] The second can 45 containing the second amplification solution 46 is similarly filled, for example, in a container 47 made of resin material and having an opening on one side, the opening of which is covered and sealed by a breakable sheet component 48.

[0441] As the breakable sheet components 43 and 48 in the first can 40 and the second can 45, laminated films such as aluminum foil or aluminum laminates are preferably used. Here, breakage refers to a state where it cannot be regenerated after breakage.

[0442] The convex deformable portions 12 and 14 at two locations on the upper shell are described in detail.

[0443] Figure 15 This is a perspective view showing the first convex deformed part 12. Figure 16 yes Figure 15 V-V' line cutting end face diagram, Figure 16 A represents the first convex deformable part 12 before deformation. Figure 16 B represents the transformed shape, and is a diagram showing the positional relationship with the first tank 40.

[0444] The first convex deformable portion 12, when pressed, moves the first can 40 to the position where the sheet component 43 is broken by the fracture portion 34 of the intermediate component 30. Specifically, the first convex deformable portion 12 is configured to be pressed downwards with a finger or the like, and by deforming the first convex deformable portion 12 in a downward convex manner (appearing as a concave portion when viewed from the outside), the first can 40 moves toward the fracture portion 34 until the sheet component 43 of the first can 40 is broken by the fracture portion 34 within the can receiving portion 32 of the intermediate component 30. Thus, the fracture portion 34 punctures the sheet component 43 of the first can 40, allowing the first amplification solution 41 to be supplied to the outside. The first amplification solution 41 is dripped from the amplification solution filling hole provided on the bottom surface of the can receiving portion 32 of the intermediate component 30 onto the upper part of the insoluble carrier 2, allowing the first amplification solution 41 to be supplied to the inspection area L1, the confirmation area L2, and the amplification index area L3 on the insoluble carrier. Additionally, at this time, the first amplification solution 41, which is dripped from the amplification solution filling hole onto the upper part of the insoluble carrier 2, fills the gap between the intermediate component 30 and the insoluble carrier 2, and is supplied through the gap to the area above the inspection area L1, the confirmation area L2 and the amplification index area L3, and slowly permeates into the insoluble carrier 2.

[0445] like Figure 16 As shown, the first convex deformable portion 12 has two protrusions 12b erected on the side facing the first can 40 at a position opposite to the first can 40. It is configured such that when the first convex deformable portion 12 is deformed by applying pressure, the two protrusions 12b abut against the first can 40 and cause the first can 40 to move.

[0446] The first convex deformable portion 12 has a centrally symmetrical mountain-shaped shape, and two protrusions 12b are symmetrically arranged with respect to the top 12a of the mountain-shaped shape, and are formed independently below (on the back side) of the inclined surface 12c that sandwiches the top 12a.

[0447] And, as Figure 16 As shown in Figure A, the first convex deformable portion 12 is formed on the upper shell 10 with two protrusions 12b symmetrically positioned relative to the center of the contact surface of the first can 40 before deformation. Furthermore, as... Figure 16 As shown by dashed lines, the fracture portion 34 of the intermediate component 30 is located below the sheet component 43 of the first can 40. When the first convex deformable portion 12 is subjected to pressure and deformed, the two protrusions 12b (the front ends of each of the two protrusions 12b) abut against the first can 40, slowly displacing towards the end side and moving the first can 40. Furthermore, as... Figure 16 As shown in B, after the deformation of the convex deformable portion 12, the distance between the two protrusions 12b increases, and the front ends of the two protrusions 12b are located further to the end side than half the distance from the center of the abutment surface of the first can 40 to the end. In this embodiment, the two protrusions 12b are provided independently, and there is a gap between the protrusions 12b (on the back side of the top 12a). The convex deformable portion 12 is formed of a soft material, thereby greatly expanding the distance between the two protrusions 12b and pressing the first can 40.

[0448] The shape or configuration of the protrusion 12b is not limited to the above-described form. For example, before deformation, the two protrusions 12b may also be positioned further to the end than half the distance from the center of the abutment surface of the first can 40 to the end.

[0449] The first convex deformable part 12, which moves the first can 40, has two protrusions 12b, thereby enabling the first can 40 to be pressed evenly at two locations, and thus enabling the first can 40 to move in parallel.

[0450] The first convex deformable portion 12 is easily deformed by pressing with a finger or the like, and becomes a downwardly convex (concave) shape. Preferably, it is configured such that the concave shape does not return to its original state after pressing, thus maintaining the state of pressing the first can 40. The first convex deformable portion 12 is configured to be pressed at the top 12a, but it can also be deformed by pressing the mountain-shaped slope using the elasticity of the convex deformable portion 12.

[0451] Figure 17 This is a perspective view showing the second convex deformed part 14. Figure 18 yes Figure 17 VII-VII' wire-cut end face view, Figure 18 A represents the second convex deformable part 14 before deformation. Figure 18 The B in the figure represents the positional relationship between the deformed part and the second can 45.

[0452] The second convex deformable portion 14 causes the sheet component 48 of the second can 45 to break by applying pressure. For example... Figure 18As shown in Figure A, the second convex deformable portion 14 has a protrusion 14b erected facing the second can 45 at a position opposite to the second can 45. Furthermore, a liquid delivery pad 4 for the test strip 1 is disposed between the second convex deformable portion 14 and the second can 45. The second convex deformable portion 14 protrudes towards the second can 45 when pressed, that is, it deforms into a concave shape when viewed from the outside, as shown in Figure A. Figure 18 As shown in Figure B, the protrusion 14b abuts against the surface of the liquid delivery pad 4, puncturing the sheet component 48 of the second tank 45, and pressing the liquid delivery pad 4 into the second tank 45. Figure 18 As shown, the second convex deformable portion 14 is configured to have a mountain-shaped top 14a on the slightly upstream side in the cross section along the upstream and downstream direction. During deformation, the protrusion 14b tilts towards the downstream side and punctures the sheet member 48.

[0453] Through this operation, the delivery pad 4 is immersed in the amplification solution 46 in the second container 45, and the second amplification solution 46 can penetrate into the delivery pad 4 and be supplied to the insoluble carrier 2 by means of capillary phenomenon.

[0454] The second convex deformable portion 14 can also be easily deformed into a concave shape by pressing with a finger or the like. Preferably, the concave shape does not return to its original state after pressing, thus maintaining the state in which the liquid delivery pad 4 is pressed into the second can 45.

[0455] This invention does not use a device connected to a power source. Instead, it deforms the first and second convex deformable portions to supply amplification fluid for highly sensitive analysis. One approach is to imagine deforming them by hand. Therefore, it is preferable to design the amplification fluid so as not to leak to the outside. Preferably, the first and second convex deformable portions 12 and 14, provided on the upper housing 10, are integrally formed seamlessly with the other parts of the upper housing 10. Preferably, the convex deformable portions 12 and 14 are made of a stretchable material and are joined to the other parts of the upper housing 10 in a sealed manner. Alternatively, the first and second convex deformable portions 12 and 14 and the other parts of the upper housing 10 can be manufactured separately and then joined together. However, it is preferable to integrally mold the first and second convex deformable portions 12 and 14 as part of the upper housing 10, as a continuous single component without any intermediate joining points, through injection molding.

[0456] The first and second convex deformable portions 12 and 14 need to have a degree of flexibility that allows them to be easily deformed with a human finger or similar means. The flexural modulus of the material constituting the convex deformable portions 12 and 14 is preferably 50 MPa or more and 350 MPa or less, more preferably 70 MPa or more and 150 MPa or less.

[0457] Furthermore, when the upper housing 10 and the lower housing 20 are simply fitted together, liquid may sometimes leak out from the gap. Therefore, it is preferable to bond the fitted portion of the upper housing 10 and the lower housing 20 in a sealed state.

[0458] As a bonding method between the upper housing 10 and the lower housing 20, ultrasonic welding is preferred. It is known that ultrasonic welding is generally difficult to weld if the parts to be welded are not made of the same material. The combination of the upper housing / lower housing is preferably polyethylene / polyethylene, polypropylene / polypropylene, or ABS (acrylonitrile-butadiene-styrene copolymer) / ABS.

[0459] On the one hand, when the convex deformable parts 12 and 14 are integrally formed on the upper shell 10, the material constituting the upper shell 10 needs to be flexible. On the other hand, in order to fix the test strip 1 or the second can 45, the lower shell 20 preferably has rigidity. Specifically, the flexural modulus of the material constituting the upper shell 10 is preferably 50 MPa or more and 350 MPa or less, more preferably 70 MPa or more and 150 MPa or less. The flexural modulus of the material constituting the lower shell 20 is preferably 500 MPa or more and 900 MPa, particularly preferably 650 MPa or more and 750 MPa or less.

[0460] In addition, the flexural modulus is the value calculated by formula (1) in accordance with the determination method of ISO 178 standard at a temperature of 20°C.

[0461] For materials used to determine the flexural modulus of elasticity, a plate-shaped test specimen with a width of b (mm) and a thickness of h (mm) is prepared and supported by two supports with a distance of L (mm) between the supports. A load F (N) is applied at the center between the supports, and the deflection (mm) in the direction of the applied load is measured. A deflection-load curve is constructed with the deflection S (mm) on the horizontal axis and the load F (N) on the vertical axis. The tangent at the origin of this curve is determined, and its slope is calculated (where (ΔF / ΔS) is the change in load ΔF (N) and the change in deflection ΔS (mm)). The flexural modulus of elasticity E (MPa) can be calculated using the following formula.

[0462] Flexural modulus E = (L 3 / (4bh 3 Equation (1) is: ))×(ΔF / ΔS)

[0463] Therefore, the combination of the upper shell and the lower shell is most preferably a combination of polypropylene / polypropylene with added softener. Here, the softener used in the polypropylene with added softener is preferably an olefin-based elastomer, and the concentration of the olefin-based elastomer relative to the polypropylene is preferably 20% by mass or more and 60% by mass or less, particularly preferably 40% by mass or more and 55% by mass or less. As a specific softener, TAFSELEN (registered trademark) manufactured by Sumitomo Chemical Co., Ltd. is an example.

[0464] Furthermore, the immunochromatographic kit of the present invention only needs to have two or more convex deformable portions. When there are three or more solutions to be supplied to the test strip, it may also have three or more convex deformable portions accordingly.

[0465] As the insoluble carrier (porous carrier) 2, a nitrocellulose membrane or the like can be used, for example. Furthermore, the back adhesive sheet 7 that fixes the insoluble carrier 2 is a sheet-like substrate with the adhesive side of the insoluble carrier 2 being adhered to it.

[0466] The marker holding pad 3 is fixed to the center of the insoluble carrier 2 along its length. The marker material can be, for example, a 50 nm diameter gold colloid (EM.GC50, manufactured by BBI). By modifying the surface of the marker material with a substance that binds to the test substance, a bond with the test substance can be formed.

[0467] The labeling material is not limited to the above; metal sulfides and coloring particles used in conventional chromatography and immunoagglutination reactions can be used, with metal colloids being particularly preferred. Examples of metal colloids include gold colloids, silver colloids, platinum colloids, iron colloids, aluminum hydroxide colloids, and composite colloids thereof. In particular, it is preferred that gold colloids show a red color and silver colloids show a yellow color within a suitable particle size, with gold colloids being the most preferred.

[0468] When the sample solution to be added is prepared by a process in which the substance to be tested, which is pre-bonded to the surface of the marker substance, is combined with the substance to be tested in the sample solution, it is preferable that the marker holding pad 3 does not contain the marker substance. In this case, the marker holding pad 3 functions as a pad indicating the position where the sample solution containing the marker substance is added.

[0469] In addition, the test strip 1 is positioned so that the opening 16 for adding the sample droplet to the upper housing 10 is aligned with the position of the mark holding pad 3.

[0470] The detection area L1 contains a second substance that binds to the test substance, and is a marker capture area that captures the marker substance that binds to the test substance. For example, when it is desired to detect influenza A virus or its biomarkers as the test substance, it is preferable to construct the detection area L1 by means of an antibody immobilization line in which an anti-influenza A monoclonal antibody (Anti-Influenza A SPTN-5 7307, manufactured by Medix Biochemica) is fixed in a linear form by physical adsorption.

[0471] When the labeled substance reaches the inspection area L1 via the complex formed by the test substance and the first substance, the second substance specifically binds to the test substance, and the labeled substance is captured via the test substance and the first substance. On the other hand, labeled substances that do not form a complex with the test substance are not captured and pass through the inspection area L1.

[0472] The confirmation region L2 contains a substance capable of binding to the first substance, which is spread from the label holding pad 3 in the insoluble carrier 2 along with the sample solution. The area where the labeled substance in the inspection region L1 is captured by the first substance is used to confirm the completion of the sample solution spread. For example, when it is desired to detect influenza A virus or its biomarkers as the test substance, it is preferable to fix the anti-mouse IgG antibody (anti-mouse IgG(H+L), rabbit F(ab')2, product number 566-70621, manufactured by FUJIFILM Wako Pure Chemical Corporation) in a linear manner, for example, by physical adsorption.

[0473] The amplification indicator region L3 contains substances that react with the second amplification solution 46. It is a region that develops color or changes color upon reaction with the second amplification solution 46, indicating the extent to which the second amplification solution 46 has spread into this region, thus serving as an indicator of the timing of the addition of the first amplification solution 41. For example, when using a mixed aqueous solution of ferric nitrate and citric acid (manufactured by FUJIFILM WakoPure Chemical Corporation, 038-06925) as the second amplification solution, it is preferable to construct the amplification indicator region L3 by immobilizing a linear line of bromocresol green (manufactured by FUJIFILM WakoPure Chemical Corporation) as a colorimetric reagent. In this case, when the second amplification solution 46 reaches the amplification indicator region L3, region L3 changes from green to orange. This color change can be used as an indicator to capture whether the inspection region L1 and the confirmation region L2 are sufficiently filled by the second amplification solution 46.

[0474] As a method for amplifying the signal of metal-based labeled substances such as metal colloids, it is preferable to use a method in which silver ions and a reducing agent for silver ions are brought into contact with the labeled substance, the silver ions are reduced by the reducing agent to generate silver particles, and the silver particles are deposited on the labeled substance with the labeled substance as the nucleus, thereby amplifying the signal of the labeled substance (hereinafter referred to as silver amplification).

[0475] To achieve silver amplification, a solution containing silver ions is used as the first amplification solution 41, and a reducing agent solution containing a reducing agent for silver ions is used as the second amplification solution 46.

[0476] (First amplification solution)

[0477] As the silver-ion-containing solution used as the first amplification solution 41, a solution in which a silver-ion-containing compound is dissolved in a solvent is preferred. As the silver-ion-containing compound, organic silver salts, inorganic silver salts, or silver complexes can be used. Inorganic silver salts or silver complexes are preferred. As the inorganic silver salt, compounds containing silver ions with high solubility in solvents such as water can be used, such as silver nitrate, silver acetate, silver lactate, silver butyrate, and silver thiosulfate. Silver nitrate is particularly preferred. As the silver complex, silver complexes coordinated to ligands having water-soluble groups such as hydroxyl and sulfone groups are preferred, such as silver hydroxysulfide.

[0478] (Second amplification solution)

[0479] As the reducing agent used in the reducing agent solution containing a reducing agent capable of reducing silver ions, which is used as the second amplification solution 46, any inorganic or organic material or mixture thereof can be used, as long as it is a substance capable of reducing silver ions to silver. As an inorganic reducing agent, Fe is a preferred example. 2+ V 2+ or Ti 3+ Reducing metal salts and reducing metal complexes are metals whose oxidation states can be altered by metal ions. When using inorganic reducing agents, it is necessary to remove or neutralize the oxidized ions by complexing or reducing them. For example, when using Fe... 2+ In systems where citric acid or EDTA (ethylenediaminetetraacetic acid) is used as a reducing agent, Fe can be formed as an oxide. 3+ The complex is made harmless. In this system, such an inorganic reducing agent is preferred, and Fe is more preferably used. 2+ Metal salts.

[0480] Alternatively, developing agents used in wet silver halide photographic materials (such as methyl gallate, hydroquinone, substituted hydroquinone, 3-pyrazolidineone, p-aminophenol, p-phenylenediamine, hindered phenol, methylamine oxime, azazine, catechol, pyrogallol, ascorbic acid (or its derivatives), and colorless dyes) and other materials that are obvious to those skilled in the art, such as those described in U.S. Patent No. 6,020,117, can also be used.

[0481] As a reducing agent, ascorbic acid reducing agents are preferred. Useful ascorbic acid reducing agents contain ascorbic acid and its analogues, isomers, and derivatives, and preferably include, for example, D- or L-ascorbic acid and its sugar derivatives (e.g., γ-lactobionic ascorbic acid, gluconic acid ascorbic acid, alginate ascorbic acid, glucoheponic acid ascorbic acid, maltobionic ascorbic acid), sodium salts of ascorbic acid, potassium salts of ascorbic acid, isoascorbic acid (or L-erythromycin ascorbic acid) and its salts (e.g., alkali metal salts, ammonium salts, or salts known in the art), enediol-type ascorbic acid, enamine-type ascorbic acid, thiol-enol-type ascorbic acid, etc., especially D, L, or D,L-ascorbic acid (and its alkali metal salts) or isoascorbic acid (or its alkali metal salts), with sodium salts being preferred. Mixtures of these reducing agents can be used as needed.

[0482] In addition, in this embodiment, the first convex deformation portion 12 moves the first can 40 toward the fracture portion 34 provided in the intermediate component 30, but the first convex deformation portion 12 can be any structure that can deform along with it and use the fracture portion 34 to break the sheet component 43 of the first can 40.

[0483] As long as the structure is such that the first amplification solution 41 flowing out of the first can 40 is broken and dripped from the amplification solution filling hole on the bottom surface of the can containing part 32 onto the insoluble carrier 2, the structure of the first can 40 and the can containing part 32 that contains the first can 40 is not limited to the structure of this embodiment.

[0484] Furthermore, having two or more protrusions in the first convex deformable portion allows the first can 40 to move parallel without tilting, which is therefore preferable. However, the first convex member may also have a single protrusion with the same shape as the second convex deformable portion in the above embodiment. A convex deformable portion with the same shape as the second convex deformable portion may also be used as the first convex deformable portion for moving the first can 40.

[0485] Figure 19 Is with Figure 18 The same cut end face view shows the shape when the first can 40 is moved using a convex deformation part 114 with the same shape as the second convex deformation part.

[0486] like Figure 19 As shown in Figure A, before deformation, the first can 40 is positioned below the protrusion 114b of the convex deformation portion 114. Furthermore, the fracture portion 34 of the intermediate component 30 is located below the first can 40. By pressing the top 114a of the convex deformation portion 114, the protrusion 114b presses against the upper surface of the first can 40, pressing the first can 40 downwards. Consequently, the fracture portion 34 punctures the sheet component 43 of the first can 40, allowing the first amplification solution 41 sealed within the first can 40 to flow out from the first can and be supplied to the test strip 1.

[0487] Thus, even if there is only one protrusion on the convex deformable part 114, the can can be moved.

[0488] In addition, the immunochromatographic kit of the present invention may also include the following: a container containing a sample extract containing an auxiliary drug for the extraction of the sample or a container containing a sample diluent, a desiccant or oxygen absorber to help preserve the kit, a user manual or other packaging instructions, and a set or part of the equipment required for the examination, such as a sample collection device such as a cotton swab.

[0489] If the immunochromatographic kit of the present invention is used, there is no need to use a special analytical device, etc., and the test can be performed with high precision using only the kit monomer.

[0490] <Immunochromatographic Examination Methods>

[0491] The immunochromatographic examination method using the above-mentioned immunochromatographic kit 100 will be briefly described.

[0492] The sample solution is dripped onto the label holding pad 3 through the sample solution dripping opening 16. If the sample solution contains the test substance, a composite of the test substance and the labeling substance is formed in the label holding pad 3 by the test substance and the first substance. This composite, along with the sample solution, expands towards the absorption pad 6 via capillary action due to the attraction of the absorption pad 6. If the composite of the test substance and the labeling substance in the sample solution is pre-formed, it is preferable that the label holding pad 3 does not contain the labeling substance, and the pre-formed composite expands towards the absorption pad 6. Simultaneously or after the dripping of the sample solution, the second convex deformable portion 14 is pressed, displacing the liquid delivery pad 4 and breaking the sheet component 48 of the second container 45, immersing the liquid delivery pad 4 in the second amplification solution 46, and conveying the second amplification solution 46 to the insoluble carrier 2. Furthermore, the timing of pressing the second convex deformable portion 14 is preferably within 30 seconds of the dripping of the sample solution, and particularly preferably immediately after the dripping of the sample solution.

[0493] The complex that reaches inspection area L1 binds to the second substance in inspection area L1 and is captured. Furthermore, the first substance, which has not bound to the tested substance, passes through inspection area L1 to confirmation area L2, where it binds to the substance bound to the first substance in confirmation area L2 and is captured.

[0494] The second amplification solution 46 passes through the inspection area L1 and the confirmation area L2 and reaches the amplification index area L3. At this time, the arrival of the second amplification solution 46 in the amplification index area L3 can be visually identified by the color change of the amplification index area L3. After confirming the color change of the amplification index area L3, the first convex deformable part 12 is pressed to supply the first amplification solution 41 onto the insoluble carrier 2.

[0495] After supplying the first amplification solution 41 to the insoluble carrier 2 and waiting for the reaction to complete, the color development of the test area L1 and the confirmation area L2 is observed through the observation window 18. The color development in test area L1 confirms the presence and concentration of the analyte, while the color development in confirmation area L2 confirms the success of the analyte assay. The color development in test area L1 and confirmation area L2 is obtained through the signal of the amplification label, enabling highly sensitive detection.

[0496] Example

[0497] The present invention will be further described in detail below through embodiments, but the present invention is not limited thereto.

[0498] [Preparation of the body fluid to be tested]

[0499] Lipoarabinomannan (LAM) (02249-61, Nacalai Tesque Inc.) (antigen) extracted from Mycobacterium tuberculosis was added to urine samples stored from healthy individuals (Bioreclamation IVT) to prepare test fluids with the LAM concentrations listed in Table 1.

[0500] [Preparation of gold colloidal pads modified with anti-lipid arabinomannan (LAM) monoclonal antibodies]

[0501] The pH was adjusted by adding 1 mL of 50 mmol / L KH₂PO₄ buffer (pH 8.0) to 9 mL of a solution containing gold colloidal particles (particle size: 50 nm) (product number: EM.GC50, manufactured by BBI). Then, 1 mL of a solution containing 20 μg / mL anti-LAM monoclonal antibody was added to the pH-adjusted solution, and the mixture was stirred for 10 minutes. Then, after standing for 10 minutes, 550 μL of an aqueous solution containing 1% by weight of polyethylene glycol (PEG; weight average molecular weight: 20000, product number: 168-11285, manufactured by FUJIFILM Wako Pure Chemical Corporation) was added and stirred for 10 minutes. Next, 1.1 mL of an aqueous solution containing 10% by weight of bovine serum albumin (BSA; Fraction V, product number: A-7906, manufactured by Sigma Corporation) was added and stirred for 10 minutes. The solution was centrifuged using a histac CF16RX centrifuge at 8000×g and 4°C for 30 minutes. 1 mL remained at the bottom of the container, and the supernatant was removed. The gold colloidal particles in the remaining 1 mL of liquid were redispersed using an ultrasonic cleaner. Then, the dispersion was placed in 20 mL of gold colloidal preservation solution (20 mmol / L). The mixture was centrifuged again under the same conditions using the same centrifuge apparatus in Tris-HCl (pH 8.2), 0.05% PEG (Mw. 20000), 150 mmol / L NaCl, and 1% BSA. The supernatant was removed, and the mixture was ultrasonically dispersed and then dispersed in gold colloidal preservation solution to obtain a solution of modified gold colloidal particles (labeled antibody) with a particle size of 50 nm modified with anti-LAM monoclonal antibody. The obtained solution was then diluted to a concentration of 20 mmol / L Tris-HCl buffer (pH 8.2) and 0.05% wt% PEG (Mw. 20000). After water extraction with sucrose concentration of 5% by mass and optical concentration of gold colloid at 520 nm of 10 mm optical path length of 0.1, 1 mL was uniformly coated onto each 5 mm × 30 cm glass fiber pad (Merck GFDX203000), and then dried in a vacuum dryer for 15 hours. The pad was then cut to obtain a pad (gold colloid holding pad) (5 mm × 4 mm) that retains modified gold colloid particles (labeled antibody) as gold colloid particles modified with anti-LAM monoclonal antibody.

[0502] [Preparation of Immunochromatographic Reagent Kits]

[0503] The immunochromatographic reagent kit is prepared as follows.

[0504] [Preparation of chromatography carriers]

[0505] As a porous carrier, a nitrocellulose membrane (with a plastic liner, HiFlowPlus HF135 (capillary flow rate = 135 s / cm, manufactured by Millipore) cut to 60 mm × 300 mm was used to form an inspection region, a confirmation region, and an amplification index region on the membrane by the following method to produce a chromatography carrier.

[0506] A 1.5 mg / mL anti-LAM antibody solution was prepared by linear coating at a position 15 mm downstream on the short side (60 mm) of the nitrocellulose membrane, serving as the examination area. A 0.5 mg / mL anti-human IgG antibody solution (anti-human IgG(H+L), rabbit F(ab')2, product number 309-006-003, manufactured by FUJIFILM Wako Pure Chemical Corporation) was prepared by linear coating at a position 11 mm downstream on the short side (60 mm), serving as the confirmation area. A 30 mmol / L bromocresol green solution (manufactured by FUJIFILM Wako Pure Chemical Corporation) was prepared by linear coating at a position 9 mm downstream on the short side (60 mm), serving as the amplification indicator area. After coating, the nitrocellulose membrane was dried at 50°C for 30 minutes using a warm air dryer. After drying, the nitrocellulose membrane, dried as described above, was immersed in 500 mL of an adhesive solution (50 mmol / L borate buffer (pH 8.5) containing 0.5% casein (derived from milk, product number 030-01505, manufactured by FUJIFILM Wako Pure Chemical Corporation)) and allowed to stand for 30 minutes. Then, the nitrocellulose membrane was removed and immersed in 500 mL of a washing / stabilizing solution (50 mmol / L Tris-HCl (pH 7.5) buffer containing 0.5% sucrose and 0.05% sodium cholate) prepared in another tank and allowed to stand for 30 minutes. The nitrocellulose membrane was then removed from the solution and dried at 25°C for 24 hours.

[0507] The portion immobilized with anti-LAM antibody corresponds to the test area containing the second substance that binds to the test substance; the portion immobilized with anti-mouse IgG antibody corresponds to the confirmation area containing the substance that can bind to the first substance; and the portion immobilized with bromocresol green corresponds to the amplification index area containing the substance that reacts with the amplification solution (reducing agent solution) sealed in the second container.

[0508] [Inspection of the preparation of test strips]

[0509] The chromatography carrier prepared as described above was attached to the back adhesive sheet (60mm × 300mm (manufactured by Adhesives Research)). Next, a 3mm wide double-sided adhesive tape (Nitto Denko Corporation) was fixed to the short side of the chromatography carrier at a position 26mm downstream. Then, the downstream end of the double-sided adhesive tape was overlapped with the downstream end of an 8mm × 300mm glass fiber pad (manufactured by Millipore), and the pre-cut pad (5mm × 30cm) without holding the gold colloid was fixed to the chromatography carrier. A delivery pad (a 25mm × 300mm glass fiber pad (manufactured by Millipore)) was attached to the upstream side of the chromatography carrier, with a 7mm overlap between the delivery pad and the chromatography carrier. The component, thus manufactured, was cut using a guillotine cutter (CM4000, manufactured by NIPPN Techno Cluster, Inc.) with a width of 5mm and parallel to the direction perpendicular to the long side of 300mm, to produce 60 test strips for examination (excluding the absorbent pad).

[0510] [Preparation of the amplification solution (reducing agent solution) to be sealed in the second container]

[0511] 23.6 mL of a 1 mol / L ferric nitrate aqueous solution (prepared by dissolving ferric nitrate(III) nonahydrate (manufactured by FUJIFILM Wako Pure Chemical Corporation, 095-00995) in water) and 13.1 g of citric acid (manufactured by FUJIFILM Wako Pure Chemical Corporation, 038-06925) were dissolved in 290 g of water. After complete dissolution, 36 mL of nitric acid (10% by weight) solution and 60.8 g of ferric ammonium sulfate(II) hexahydrate (manufactured by FUJIFILM Wako Pure Chemical Corporation, 091-00855) were added while stirring. This solution was used as the second amplification solution, i.e., the reducing agent solution, sealed in the second tank.

[0512] [Preparation of the amplification solution (silver ion solution) to be sealed in the first container]

[0513] 8 mL of silver nitrate solution (containing 10 g of silver nitrate) and 24 mL of 1 mol / L ferric nitrate aqueous solution were added to 66 g of water. Furthermore, this solution was mixed with 5.9 mL of nitric acid (10 wt%), 0.1 g of dodecaneamine (manufactured by FUJIFILM Wako Pure Chemical Corporation, 123-00246), and surfactant C. 12 H 25 -C6H4-O-(CH2CH2O) 50 A solution of 0.1g of H dissolved in 47.6g of water was mixed, and this mixture was used as the first amplification solution, namely the silver ion solution, to be sealed in the first tank.

[0514] [Making Absorbent Pads]

[0515] Prepare 60 glass fiber pads (glass filter paper, manufactured by Advantech Co., Ltd.) cut into 12mm×10mm pieces to serve as absorbent pads.

[0516] [Preparation of Immunochromatographic Reagent Kit Components]

[0517] Using polypropylene as the material, injection molding is used to produce products such as... Figures 12-14The diagram shows the lower shell 20, upper shell 10, intermediate component 30, and first tank 40 and second tank 45 constituting the immunochromatographic reagent kit 100. The upper shell is manufactured by injection molding using polypropylene containing 50% by mass of TAFSELEN (a registered trademark), an olefin-based elastomer manufactured by Sumitomo Chemical Co., Ltd. Furthermore, the upper shell 10 has two deformable portions (a first convex deformable portion and a second convex deformable portion), which are not separated from the upper shell 10 and are formed as part of the upper shell 10 at all boundaries by injection molding.

[0518] In addition, the upper housing in the embodiment is configured with the following structure, namely, Figure 12 and Figure 13 The first convex deformable portion 12 shown has two protrusions, and the second convex deformable portion 14 has one protrusion.

[0519] [Preparation of Immunochromatographic Reagent Kits]

[0520] like Figures 12-14 The lower housing 20, the test strip 1 manufactured as described above, and the absorbent pad 6 manufactured as described above are fixed in place. Next, the first amplification solution 41 sealed in the first canister 40 and the second amplification solution 46 sealed in the second canister 45, manufactured as described above, are respectively filled into the first canister 40 and the second canister 45. The second canister 45 is sealed with aluminum foil as a sheet component 48, and the first canister 40 is sealed with aluminum foil as a sheet component 43, as shown. Figures 12-14 As shown, the second can 45 is mounted on the lower housing 20 with the sheet component 48 positioned on top, and the first can 40 is mounted on the middle component 30 with the sheet component 43 positioned below. Furthermore, with the upper housing 10 and the lower housing 20 interlocked at their outer peripheries, the contact portions of the upper and lower housings are joined by ultrasonic welding. At this point, it is confirmed that the welded areas are uniformly welded in a sealed state across all areas. This is how an immunochromatographic reagent kit is manufactured.

[0521] [Example 1]

[0522] [Fabrication of Concentration Devices]

[0523] To produce Figure 5 and Figure 9 The concentration device 201 is shown. Additionally... Figure 9 This is a perspective view of a portion (upper part) of the condenser 201. (See image below.) Figure 9 As shown, the concentration device 201 of Example 1 has a cover 280 with a recovery port 282.

[0524] Specifically, in cylinder 211 (12 mm inner diameter, 60 mm depth, cylindrical shape, with external threads on the top), 50 μL of an 800 mmol / L Tricine buffer (pH 8.5) (347-02844, manufactured by FUJIFILM Wako Pure Chemical Corporation) containing 0.2% by mass casein (030-01505, manufactured by FUJIFILM Wako Pure Chemical Corporation) and 2% by mass Tween40 (T2531, manufactured by Tokyo Chemical Industry Co., Ltd.) was added. The solution was dried for 3 days at 25°C and below 10% Rh (relative humidity), followed by vacuum drying for 24 hours to solidify it, thus producing a drying reagent. Furthermore, one gold colloidal retaining pad (5 mm × 4 mm) and 700 mg of superabsorbent polymer 230 (described later) were added.

[0525] Furthermore, a piston 220 with a front end 221 having holes 222 (1 mm in diameter and 24 holes) smaller than the water-absorbing particle size of the superabsorbent polymer 230 and a cap 280 (with internal threads) having a hose 281 with a retraction port 282 is prepared and combined with the cylinder 211 to complete the concentration device 201.

[0526] <Superabsorbent polymer>

[0527] 700 mg of commercially available superabsorbent polymer particles (manufactured by M2Polymer Technologies Inc.; SAP Sphere 2.5 mm) were granulated to prepare superabsorbent polymer 230 used in the examples. Superabsorbent polymer 230 has a particle size of 2.5 mm, a swelling ratio of 13 g / g, and a water absorption rate of 0.5 g / min.

[0528] [Concentration of the tested body fluids]

[0529] Using the obtained concentrator 201, the above-mentioned body fluid was concentrated as shown in Figure 1.

[0530] <Injection procedure of the tested body fluid>

[0531] First, remove the cap 280 and piston 220 from the concentrator 201. Then, inject 4.5 mL of the tested body fluid into the cylinder 211 through the opening 216 and stir. Figure 1B ).

[0532] <Water Absorption Process>

[0533] Then, the concentrating device 201 is left to stand for 60 minutes. During this period, the water contained in the test fluid 240 is almost completely absorbed by the superabsorbent polymer 230, generating a test fluid concentrate 246, which is a concentrate of the test fluid 240 (the superabsorbent polymer 230 becomes a swollen superabsorbent polymer 232). Figure 1C Furthermore, as described above, since the cylinder 211 contains a gold colloidal holding pad, an antigen-antibody reaction occurs simultaneously with concentration, forming a gold particle complex, which is a complex of LAM contained in the test fluid and gold colloidal particles modified with anti-LAM monoclonal antibody contained in the cylinder, i.e., modified gold colloidal particles (labeled antibody). That is, in the obtained test fluid concentrate 246, LAM forms a gold particle complex as a complex with modified gold colloidal particles (labeled antibody).

[0534] <Extract Addition Process>

[0535] Next, 400 μL of extraction buffer 250 (PBS(-) manufactured by FUJIFILM Wako PureChemical Corporation 166-23555) was added to the concentrated body fluid sample 246. Figure 1D ).

[0536] <Removal Process>

[0537] Furthermore, the piston 220 is inserted into the cylinder 211 through the opening 216, and the cap 280 is tightened from above the piston 220. By tightening the cap 280 with threads, the piston 220 is pressed downwards, and a concentrated body fluid solution 248 (as a concentrated solution of the tested body fluid) is obtained through the hole 222 at the front end 221 of the piston 220. Figure 1E The concentrator 201 is reversed, causing the obtained concentrated body fluid 248 to move to the tubing 281. The concentrated body fluid 248 is then removed from the recovery port 282 by pressing the tubing 281. Furthermore, as described above, similar to the concentrated body fluid 246 obtained in the water absorption process, LAM forms a gold particle complex in the concentrated body fluid 248.

[0538] [LAM detection]

[0539] The obtained concentrated body fluid solution 248 (24 μL) is added dropwise to the immunochromatographic kit prepared as described above. Immediately after addition, the second convex deformable part 14 is pressed, thereby breaking the sheet component 48, i.e., the aluminum foil, that seals the second amplification solution 46 sealed in the second container 45. The delivery pad 4 is then immersed in the second container 45, thereby supplying the second amplification solution 46 to the porous carrier 2 by means of capillary action.

[0540] After the amplification index region L3 changes from green to orange, the first convex deformation part 12 is pressed, causing the first can 40 to move towards the fracture part 34 of the can receiving part 32 of the intermediate component 30. This fracture part 34 then breaks the sheet component 43 (aluminum foil) sealing the first can 40, allowing the first amplification solution 41 (silver ion solution) to be supplied to the porous carrier 2 through the opening of the intermediate component 30, and the silver amplification reaction occurs. The silver amplification reaction ends within tens of seconds.

[0541] After the silver amplification reaction was completed, the staining was visually confirmed. The results are shown in Table 1.

[0542] +: Colored

[0543] -: Uncolored

[0544] [Example 2]

[0545] [Fabrication of Concentration Devices]

[0546] To produce Figure 6 and Figure 9 The concentration device 202 shown is identical to the concentration device 201 of Example 1, except that the cylinder 211 is replaced by the cylinder 212.

[0547] Specifically, cylinder 212 (12mm inner diameter, 60mm depth, cylindrical shape, with external threads on the upper part) is used instead of cylinder 211. Otherwise, the concentrator (concentrator 202) is manufactured according to the same steps as in Example 1.

[0548] Here, the cylinder 212 has a partition wall 260 at a position 3.5 mm from the bottom surface 218. This partition wall 260 is disposed on the inner circumferential surface of the cylinder 212 in a manner that allows it to move along the length of the cylinder 212. The partition wall 260 has pores 262 (pore diameter 1 mm, number of pores 24) smaller than the particle size of the superabsorbent polymer 230 before water absorption. The portion (400 μL) surrounded by the bottom 217 and the partition wall 260 corresponds to the extract holding portion described above. The partition wall 260 is positioned by a protrusion (not shown) formed on the inner circumferential surface at a position 3.5 mm from the bottom surface of the cylinder 212, but by applying pressure from above, it can penetrate the extract holding portion beyond the protrusion.

[0549] In addition, such as Figure 6 As shown, in the concentrator 202, the superabsorbent polymer 230 is contained in the cylinder 212 above and in contact with the partition wall 260.

[0550] [Concentration of the tested body fluids]

[0551] Using the obtained concentrator 202, the above-mentioned body fluid was concentrated as shown in Figure 2.

[0552] <Injection procedure of the tested body fluid>

[0553] First, remove the cap 280 and piston 220 from the concentrator 202. Then, inject 4.5 mL of the test fluid into the cylinder 212 through the opening 216 and stir. At this time, a portion of the test fluid (test fluid 241: 400 μL) is introduced into the space below the partition 260 through the hole 262. Figure 2B ).

[0554] <Water Absorption Process>

[0555] Then, the concentrator 202 is left to stand for 60 minutes. During this period, the water contained in the test fluid 242 (the test fluid 242 other than the test fluid 241 held as an extractant) existing only above the partition 260 in the test fluid 240 is almost completely absorbed by the superabsorbent polymer 230, and a test fluid concentrate 246 is generated in the cylinder 212 as a concentrate of the test fluid 242 (the superabsorbent polymer 230 becomes a swollen superabsorbent polymer 232). Figure 2C Additionally, similar to Example 1, in the obtained test fluid concentrate 246, LAM forms a gold particle complex as a complex with modified gold colloidal particles (labeled antibody).

[0556] <Extract Addition Process>

[0557] Next, the piston 220 is inserted into the cylinder 212 through the opening 216, and the superabsorbent polymer 232 is pressed downwards, thereby moving the partition wall 260 to the bottom surface 218 of the cylinder 212. The test fluid 241 held in the extraction solution holding section is then introduced through the hole 262 of the partition wall 260 to the area above the partition wall 260. Thus, the test fluid 241 held in the extraction solution holding section is added to the test fluid concentrate 246. Figure 2D ).

[0558] <Removal Process>

[0559] Furthermore, following the same steps as in Example 1, the concentrated body fluid 248 was removed.

[0560] [LAM detection]

[0561] The LAM was measured in the obtained concentrated body fluid solution 248 (24 μL) following the same procedure as in Example 1. The results are shown in Table 1.

[0562] [Example 3]

[0563] [Fabrication of Concentration Devices]

[0564] To produce Figure 7 and Figure 9 The concentrator 203 shown is identical to the concentrator 201 of Example 1, except that the cylinder 211 is replaced by the cylinder 213.

[0565] Specifically, cylinder 211 is replaced by cylinder 213 (12mm inner diameter, 60mm depth, cylindrical shape, with external threads on the upper part). Otherwise, the concentrator (concentrator 203) is manufactured according to the same steps as in Example 1.

[0566] Here, a porous synthetic resin 270 (a sponge made of PVA (polyvinyl alcohol)) (90% porosity) is contained at the bottom 217 (4 mm from the bottom surface 218) of the cylinder 213. The pores (not shown) of the synthetic resin 270 are smaller than the particle size of the superabsorbent polymer 230 before water absorption. The pores of the synthetic resin 270 correspond to the extract holding section described above.

[0567] In addition, such as Figure 7 As shown, in the concentrator 203, the superabsorbent polymer 230 is contained in the cylinder 213 above and in contact with the synthetic resin 270.

[0568] [Concentration of the tested body fluids]

[0569] Using the obtained concentrator 203, the above-mentioned body fluid was concentrated as shown in Figure 3.

[0570] <Injection procedure of the tested body fluid>

[0571] First, remove the cap 280 and piston 220 from the concentrator 203. Then, inject 4.5 mL of the test fluid into the cylinder 213 through the opening 216 and stir. At this time, a portion of the test fluid (test fluid 241: 400 μL) is introduced into the pores of the synthetic resin 270 (synthetic resin 270 becomes synthetic resin 272 into the pores of test fluid 241, which is part of test fluid 240). Figure 3B ).

[0572] <Water Absorption Process>

[0573] Then, the concentrator 203 is allowed to stand for 60 minutes. During this period, the water contained in the test fluid 242 (the test fluid 242 other than the test fluid 241 held as an extractant) which exists only above the synthetic resin 270 in the test fluid 240 is almost completely absorbed by the superabsorbent polymer 230, and a test fluid concentrate 246 is generated in the cylinder 213 as a concentrate of the test fluid 242 (the superabsorbent polymer 230 becomes a swollen superabsorbent polymer 232). Figure 3CAdditionally, similar to Example 1, in the obtained test fluid concentrate 246, LAM forms a gold particle complex as a complex with modified gold colloidal particles (labeled antibody).

[0574] <Extract Addition Process>

[0575] Next, the piston 220 is inserted into the cylinder 213 through the opening 216, pressing the superabsorbent polymer 232 downwards, thereby crushing the synthetic resin 270, and introducing the test fluid 241 held in the extract holding section through the holes of the synthetic resin 270 to the top of the synthetic resin 270. Thus, the test fluid 241 held in the extract holding section is added to the test fluid concentrate 246. Figure 3D ).

[0576] <Removal Process>

[0577] Furthermore, following the same steps as in Example 1, the concentrated body fluid 248 was removed.

[0578] [LAM detection]

[0579] The LAM was measured in the obtained concentrated body fluid solution 248 (24 μL) following the same procedure as in Example 1. The results are shown in Table 1.

[0580] [Example 4]

[0581] [Fabrication of Concentration Devices]

[0582] To produce Figure 8 and Figures 9-10 The concentrator 204 shown is identical to the concentrator 201 of Example 1, except that the cylinder 211 is replaced by the cylinder 214 and the piston 220 is replaced by the piston 224.

[0583] Specifically, cylinder 211 is replaced by cylinder 214 (12mm inner diameter, 60mm depth, cylindrical shape, with external threads on the upper part) and piston 220 is replaced by piston 224. Otherwise, the concentrator (concentrator 204) is manufactured according to the same steps as in Example 1.

[0584] Here, the cylinder 214 and piston 224 are equipped with a piston position fixing mechanism, which overcomes the pressure of the water absorption and expansion of the superabsorbent polymer 230 and fixes the front end 221 of the piston 224 at the above-mentioned position A (specifically, in the test fluid injection process described later, the position 3.5 mm lower than the liquid level 244 of the test fluid 240 injected into the cylinder 214).

[0585] More specifically, such as Figure 10As shown, the cylinder 214 has a cutout 215, and the piston 224 has a protrusion 223. The piston 224 is inserted into the cylinder 214, and the protrusion 223 of the piston 224 is hung on the cutout 215 of the cylinder 214, thereby overcoming the pressure of water absorption and expansion accompanying the superabsorbent polymer 230, and fixing the front end 221 of the piston 224 at position A.

[0586] [Concentration of the tested body fluids]

[0587] Using the obtained concentrator 204, the above-mentioned body fluid was concentrated as shown in Figure 4.

[0588] <Injection procedure of the tested body fluid>

[0589] First, remove the cap 280 and piston 220 from the concentrator 204. Then, inject 4.5 mL of the test fluid into the cylinder 214 through the opening 216, stir, and insert the piston 224 into the cylinder 214, hooking the protrusion 223 of the piston 224 onto the cut 215 of the cylinder 214, thereby fixing the front end 221 of the piston 224 at the aforementioned position A. At this time, a portion of the test fluid (test fluid 241: 400 μL) is introduced through the hole 222 of the front end 221 of the piston 224 above the front end 221 of the piston 224. Figure 4B ).

[0590] <Water Absorption Process>

[0591] Then, the concentrator 204 is left to stand for 60 minutes. During this period, the water contained in the body fluid 242 (the body fluid 242 in the body fluid 240 other than the body fluid 241 held as extractant) which exists only below the front end 221 of the piston 224 in the body fluid 240 is almost completely absorbed by the superabsorbent polymer 230, and a body fluid concentrate 246 is generated in the cylinder 214 as a concentrate of the body fluid 242 (the superabsorbent polymer 230 becomes a swollen superabsorbent polymer 232). Figure 4C Additionally, similar to Example 1, in the obtained test fluid concentrate 246, LAM forms a gold particle complex as a complex with modified gold colloidal particles (labeled antibody).

[0592] <Extract Addition Process>

[0593] Next, the piston 224 is pulled up, and the test fluid 241 existing above the front end 221 of the piston 224 is introduced through the hole 222 of the front end 221 of the piston 224 to below the front end 221 of the piston 224, thereby adding the test fluid 241 existing above the front end 221 of the piston 224 to the test fluid concentrate 246. Figure 4D ).

[0594] <Removal Process>

[0595] Furthermore, following the same steps as in Example 1, the concentrated body fluid 248 was removed.

[0596] [LAM detection]

[0597] The LAM was measured in the obtained concentrated body fluid solution 248 (24 μL) following the same procedure as in Example 1. The results are shown in Table 1.

[0598] [Example 5]

[0599] Add 300 mg of superabsorbent polymer 230 to cylinder 211, and otherwise prepare a concentration device 201 following the same steps as in Example 1.

[0600] Except for using the obtained concentration device 201, the concentrate of the test sample was taken out and LAM was detected following the same procedure as in Example 1. The results are shown in Table 1.

[0601] [Comparative Example 1]

[0602] The superabsorbent polymer 230 was not added to the cylinder 211. Otherwise, the concentration device (comparative device 1) was prepared following the same steps as in Example 1.

[0603] Except for using the obtained comparison device 1, when the test body fluid was concentrated according to the same procedure as in Example 1, it was not concentrated, and the test body fluid itself was taken out. Furthermore, the taken out test body fluid was subjected to LAM detection according to the same procedure as in Example 1. The results are shown in Table 1.

[0604] [Comparative Example 2]

[0605] Similar to Example 1, the sample fluid injection process, water absorption process, and extractant addition process were performed. However, when the cylinder 211 was tilted without using the piston 220 to remove the sample fluid concentrate, it was impossible to remove the sample fluid concentrate.

[0606] [Comparative Example 3]

[0607] Similar to Example 1, the injection and aspiration processes of the tested body fluid were performed. Then, when no extractant was added and the removal process was performed in the same manner as in Example 1, the concentrated body fluid (concentrated body fluid) could not be removed.

[0608] [Table 1]

[0609]

[0610] When using the methods of Examples 1 to 5, which are concentration methods of the present invention, a concentrated solution of the test body fluid at the desired concentration ratio can be obtained. On the other hand, in Comparative Example 1, which did not use a superabsorbent polymer, the test body fluid could not be concentrated. Furthermore, in Comparative Example 2, which did not use a specified piston, the concentrated solution of the test body fluid could not be extracted. Furthermore, in Comparative Example 3, which did not add an extractant, the concentrated solution of the test body fluid (test body fluid concentrate) could not be extracted either.

[0611] Symbol Explanation

[0612] 1. Test strips for inspection

[0613] 2. Insoluble carrier (porous carrier)

[0614] 3. Marking retaining pad (fiberglass pad)

[0615] 4 pad for liquid delivery

[0616] 6 Absorbent Pads

[0617] 7. Back adhesive sheet

[0618] 9. Outer shell

[0619] 10 Upper shell

[0620] 12 First convex deformation part

[0621] 12a Top of the first convex deformed part

[0622] 12b The protrusion of the first convex deformed part

[0623] 12c Inclined surface of the first convex deformed part

[0624] 14 Second convex deformation part

[0625] 14a Top of the second convex deformation part

[0626] 14b The protrusion of the second convex deformed part

[0627] 16. Sample droplet addition using an opening

[0628] 18 Observation Window

[0629] 20 Lower shell

[0630] 21. Insoluble carrier container (porous carrier container)

[0631] 22 Absorbent Pad Receptacle

[0632] 24. Second tank container section

[0633] 30 Intermediate components

[0634] 32 First tank containing section

[0635] 34. Fracture section

[0636] 35 Flow path forming part

[0637] 36 Back surface of flow path forming part 35

[0638] 40. First amplification solution for first container.

[0639] 41 First Amplification Solution

[0640] 42 containers

[0641] 43 Sheet components

[0642] 45. Use the second amplification solution in the second container.

[0643] 46 Second amplification solution

[0644] 47 containers

[0645] 48 Sheet components

[0646] 100 Immunochromatographic Reagent Kit

[0647] 114 Convex Deformation Section

[0648] 114a Top of the convex deformed part 114

[0649] 114b The protrusion of the convex deformed part 114

[0650] 201, 202, 203, 204 Concentrated Devices

[0651] Cylinder blocks 211, 212, 213, and 214

[0652] 215 Incision

[0653] 216 Opening

[0654] 217 Bottom

[0655] 218 Bottom

[0656] 220 and 224 pistons

[0657] 221 Front end

[0658] 222 Hole at the front end

[0659] 223 Protrusion

[0660] 230 Superabsorbent polymer (Superabsorbent polymer before water absorption)

[0661] 232 Superabsorbent polymers (superabsorbent polymers after absorbing water) (swelling superabsorbent polymers)

[0662] 240, 241, 242 Sampled bodily fluids

[0663] 244. The level of the body fluid being examined.

[0664] 246. Concentrated body fluids from the test subjects

[0665] 248. Concentrated body fluid solution from the test subject

[0666] 250 extract

[0667] 260 next door

[0668] 262 The hole next door

[0669] 270 Synthetic Resin

[0670] 272 The sample fluid was introduced into the synthetic resin in the well.

[0671] 280 Cover with recycling port

[0672] 281 Hose

[0673] 282 Recycling Port

[0674] 300 Nitrocellulose membrane

[0675] 301 Gold Colloidal Retention Pad

[0676] 302 test line

[0677] 303 Control Line

[0678] 304 Immobilization line for colorimetric reagent

Claims

1. A method of concentrating a test body fluid, comprising, in order: a test body fluid injection step of injecting a test body fluid, which is an aqueous solution containing a high molecule, into a cylinder body in which a particulate superabsorbent polymer is housed; a water absorption step of absorbing water contained in the test body fluid injected into the cylinder body by the superabsorbent polymer housed in the cylinder body to generate a test body fluid concentrate, which is a concentrate of the test body fluid, in the cylinder body; a liquid addition step of adding less liquid to the test body fluid concentrate than the test body fluid injected into the cylinder body in the test body fluid injection step; and a withdrawal step of inserting a piston, which is capable of being inserted into the cylinder body and has a front end portion having a pore smaller than a particle diameter of the superabsorbent polymer after water absorption, into the cylinder body to withdraw a test body fluid concentrate, which is a concentrated liquid of the test body fluid, through a pore of the front end portion of the piston.

2. The method of concentrating a test body fluid according to claim 1, wherein the test body fluid injection step is a step of injecting the test body fluid into the cylinder body while retaining a part of the test body fluid injected into the cylinder body as the liquid to be added in the liquid addition step in the cylinder body, the water absorption step is a step of absorbing water contained in the test body fluid injected into the cylinder body except for the test body fluid retained as the liquid to be added in the liquid addition step by the superabsorbent polymer housed in the cylinder body to generate the test body fluid concentrate in the cylinder body, the liquid addition step is a step of adding the test body fluid retained as the liquid to be added in the liquid addition step to the test body fluid concentrate.

3. The method of concentrating a test body fluid according to claim 2, wherein the cylinder body has a liquid retaining portion for retaining the liquid to be added in the liquid addition step at a bottom portion, and the superabsorbent polymer is housed in the cylinder body in contact with the liquid retaining portion above the liquid retaining portion, the test body fluid injection step is a step of injecting the test body fluid into the cylinder body while retaining a part of the test body fluid injected into the cylinder body as the liquid to be added in the liquid addition step in the liquid retaining portion.

4. The method of concentrating a test body fluid according to claim 3, wherein the liquid retaining portion is a portion surrounded by the bottom portion of the cylinder body and a partition wall provided on an inner peripheral surface of the cylinder body in a manner capable of moving in a length direction of the cylinder body, the partition wall having a pore smaller than a particle diameter of the superabsorbent polymer before water absorption, the liquid addition step is a step of moving the partition wall to a bottom surface of the cylinder body to introduce the test body fluid retained in the liquid retaining portion above the partition wall through the pore of the partition wall, thereby adding the test body fluid retained in the liquid retaining portion to the test body fluid concentrate.

5. The method of concentrating a test body fluid according to claim 3, wherein ​ The liquid holding portion is a portion formed by a hole of a porous resin housed in the bottom of the cylinder, the hole of the resin being smaller than the particle diameter of the superabsorbent polymer before water absorption, The liquid adding step is a step of crushing the resin and introducing the body fluid held in the liquid holding portion through the hole of the resin to above the resin, thereby adding the body fluid held in the liquid holding portion to the body fluid concentrate.

6. The method of concentrating a body fluid according to claim 5, wherein The porous resin is a sponge.

7. The method of concentrating a body fluid according to claim 2, wherein The body fluid injecting step is a step of injecting the body fluid into the cylinder while inserting the piston into the cylinder and fixing the front end portion of the piston to a position lower than the liquid surface of the body fluid injected into the cylinder and higher than the superabsorbent polymer housed in the cylinder, thereby holding the body fluid present above the front end portion of the piston among the body fluid injected into the cylinder as the liquid added in the liquid adding step, The liquid adding step is a step of pulling up the piston and introducing the body fluid present above the front end portion of the piston through the hole of the front end portion of the piston to below the front end portion of the piston, thereby adding the body fluid present above the front end portion of the piston to the body fluid concentrate.

8. The method of concentrating a body fluid according to claim 1 or 2, wherein In the taking-out step, the taken-out body fluid concentrate is further recovered using a lid having a recovery port for recovering the body fluid concentrate.

9. The method of concentrating a body fluid according to claim 1 or 2, wherein The water absorption speed of the superabsorbent polymer is 0.01 g / min or more and 40 g / min or less per 1 g of the superabsorbent polymer.

10. The method of concentrating a body fluid according to claim 1 or 2, wherein The particle diameter of the superabsorbent polymer is 5 mm or less.

11. The method of concentrating a body fluid according to claim 1 or 2, wherein The swelling ratio of the superabsorbent polymer is greater than 0.2 g / g and less than 800 g / g.

12. The method of concentrating a body fluid according to claim 1, wherein The body fluid is an aqueous solution containing a high molecule contained in a biological fluid.

13. The method of concentrating a body fluid according to claim 12, wherein The cylinder further contains a binding substance that specifically binds to the high molecule contained in the biological fluid.

14. The method of concentrating a body fluid according to claim 13, wherein The binding substance is contained in the cylinder as a complex with a metal particle.

15. The method of concentrating a body fluid according to claim 13 or 14, wherein The high molecule contained in the biological fluid is an antigen, and the binding substance is an antibody against the antigen.

16. The method of concentrating a body fluid according to claim 1 or 2, wherein The cylinder further contains at least one selected from the group consisting of casein and tris(hydroxymethyl)methylglycine.

17. The method of concentrating a subject fluid according to claim 1 or 2, wherein, the subject fluid is urine.

18. A method of examining a subject fluid that detects a high molecule in a subject fluid that is an aqueous solution containing a high molecule, the method of examining a subject fluid successively comprises: a concentrating step of obtaining a subject fluid concentrate using the method of concentrating a subject fluid according to any one of claims 1 to 17; and a detecting step of detecting a high molecule in the obtained subject fluid concentrate.

19. The method of examining a subject fluid according to claim 18, wherein, the subject fluid is an aqueous solution containing an antigen or not containing an antigen, the concentrating step is a step of obtaining an antigen concentrate by concentrating the aqueous solution containing the antigen or not containing the antigen using the method of concentrating a subject fluid according to any one of claims 1 to 17, the detecting step is a step of detecting the antigen in the antigen concentrate by an immunochromatography method using an antigen-antibody reaction.

20. The method of examining according to claim 19, wherein, the detecting step comprises an amplifying step of amplifying information of the antigen in the antigen concentrate.

21. The method of examining according to claim 20, wherein, the amplifying step is a silver amplifying step.

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