Antigen detection system, antigen detection kit, and antigen detection method

Abstract

To provide an accurate and quick antigen detection system capable of performing accurate quantitative analysis and shortening the detection time by a simple operation.SOLUTION: Disclosed is an antigen detection system using an enzyme-labeled antibody reactive with antigen which includes: an introduction unit which may contain the enzyme-labeled antibody; a purification unit to which the antigen analog reacting with the enzyme-labeled antibody is fixed; a reaction unit to which a substrate reacting with enzyme of the enzyme-labeled antibody is fixed: and a detection unit for detecting an increase or decrease in an electrical signal.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an antigen detection system that utilizes an antigen-antibody reaction. [Background technology]

[0002] In point-of-care testing (POCT), immunochromatography (also known as lateral flow immunoassay), which uses antigen-antibody reactions as its detection principle, is widely used. However, it is not suitable for quantitative measurement due to variations in color development resulting from the non-uniformity of the colored particles used and its weak color development.

[0003] In the medical testing industry, which uses POCT test kits, there is a need for quantitative measurements, and various methods for quantification are being attempted.

[0004] However, in order to achieve stable quantitative measurements with immunochromatography, it is necessary to develop materials with high signal reproducibility. In particular, colored particles, such as gold colloid particles and colored latex particles, are used as carriers to support antibodies, etc. These carriers have many technical challenges, such as the requirement for uniform particle size to obtain stable and reproducible color signals. Currently, quantitative measurements using gold colloid particles are being attempted in the widely used immunochromatography kits, which optically read the degree of coloration of the detection line or detection surface, but reproducible quantitative detection is difficult.

[0005] Instead of optical detection, electrochemical detection methods are expected to stabilize the signal by detecting an electrical signal, making it possible to perform highly sensitive measurements.

[0006] In electrochemical detection, there is a method in which the electrode surface is modified and changes in the properties of the electrode that occur when the target substance is captured are detected, but stable detection is difficult because the degree of modification of the electrode surface is non-uniform. A method that directly detects electrical signals without modifying the electrode is thought to be suitable for stable detection. An "electrochemical immunochromatography method" has been proposed that combines a simple immunochromatography method with an electrochemical method using an enzyme reaction that is suitable for stable detection (Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 6366047 [Patent Document 2] Patent No. 5097557 Summary of the Invention [Problem to be solved by the invention]

[0008] However, the above method requires at least three steps from the introduction of the sample to the detection of the target substance, and takes about 15 minutes. When applying this method to a POCT kit for healthcare use, the number of steps is large, the operation is complicated, and it takes a long time to detect, making it inconvenient for general end users to use.

[0009] The present invention has been made in consideration of the above-mentioned problems, and an object of the present invention is to provide an accurate and rapid antigen detection system that is capable of accurate quantitative analysis and can shorten the detection time with simple operations. [Means for solving the problem]

[0010] As a result of extensive research, the inventors of the present invention came up with the idea that an antigen detection system having four different units can be configured as a device to perform antigen detection measurement that realizes accurate and rapid quantitative analysis, and thus completed the present invention. The present invention includes the following.

[0011] [1] An antigen detection system using an enzyme-labeled antibody that reacts with an antigen, A transfer unit which may include an enzyme-labeled antibody; a purification unit on which an antigen analogue that reacts with the enzyme-labeled antibody is immobilized; a reaction unit having a substrate immobilized thereon that reacts with the enzyme of the enzyme-labeled antibody; and a detection unit that detects an increase or decrease in an electrical signal. [2] The antigen detection system described in [1], wherein the reaction unit and the detection unit are present on the same flow path. [3] The antigen detection system according to [1] or [2], wherein the introduction unit, the purification unit, the reaction unit and the detection unit are configured so that the sample liquid can move in this order. [4] The antigen detection system according to any one of [1] to [3], wherein the enzyme of the enzyme-labeled antibody is at least one selected from the group consisting of hydrolases and oxidoreductases. [5] The antigen detection system described in any one of [1] to [4], wherein the support to which the antigen analogue is fixed in the purification unit is at least one selected from the group consisting of a membrane filter, an immunochromatographic membrane, a nonwoven fabric, a filter paper, a mesh, and a carrier microparticle. [6] The antigen detection system according to any one of [1] to [5], wherein the detection unit has an electrode including at least a working electrode and a counter electrode. [7] An antigen detection kit comprising the antigen detection system according to any one of [1] to [6] immobilized on a support, and an enzyme-labeled antibody. [8] An antigen detection method using the antigen detection system according to any one of [1] to [7], (1) a first step of contacting a sample containing an antigen to be detected with an enzyme-labeled antibody to obtain a test sample; (2) a second step of contacting the test sample obtained in the first step with a purification unit to capture unreacted enzyme-labeled antibodies in the purification unit to obtain a purified sample; (3) a third step of contacting the purified sample with a reaction unit to react the enzyme of the enzyme-labeled antibody with a substrate; and (4) A fourth step of detecting an electric signal generated by contacting the reaction product of the third step with a detection unit. A method for detecting an antigen, comprising: Effect of the Invention

[0012] The antigen detection system of the present invention enables rapid and highly sensitive quantitative measurement with simple operations. In particular, by providing a purification unit capable of capturing free enzyme-labeled antibodies that are not bound to antigens, the number of steps required for operation is reduced, making it possible to measure the amount of the antigen to be detected in a shorter time. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram (front view) of an antigen detection system according to one embodiment of the present invention. [Diagram 2] FIG. 1 is a schematic diagram (side view) of an antigen detection system according to one embodiment of the present invention. [Diagram 3] FIG. 2 is a schematic diagram of an example of a detection gold electrode used in the present invention. [Figure 4] 1 shows detection results obtained with an antigen detection system according to one embodiment of the present invention. [Diagram 5] 1 shows detection results obtained with an antigen detection system according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will now be described in further detail.

[0015] <Antigen detection system> The antigen detection system is an immunoassay system capable of measuring the amount of the antigen to be detected by specifically binding the antigen to be detected in a sample with an enzyme-labeled antibody and measuring the amount of the substrate that reacts with the labeling enzyme in the binding body, and has the following four units and is configured so that the sample liquid can move through each unit in order. Here, the sample liquid refers to all liquids that move through the antigen detection system. The first site is the "introduction unit" where the sample and enzyme-labeled antibody are introduced, the second site is the "purification unit" which captures free enzyme-labeled antibody in the test sample, the third site is the "reaction unit" where the labeled enzyme reacts with the substrate, and the fourth site is the "detection unit" which detects the current using an electrode. In summary, it is composed of four units.

[0016] The antigen detection system is preferably configured so that a sample or a test sample mixed with an enzyme-labeled antibody passes through an introduction unit, a purification unit, a reaction unit, and a detection unit in this order.

[0017] The introduction unit is a unit that is provided at the most upstream position in the flow of the sample liquid and introduces the sample into the system. The antigen to be detected contained in the sample may be bound to an enzyme-labeled antibody in advance and then applied to the introduction unit, or the introduction unit may contain an enzyme-labeled antibody. In this case, the sample can be applied directly to the introduction unit. In either case, the sample is brought into contact with an excess amount of the enzyme-labeled antibody to prepare a test sample.

[0018] In the purification unit, the enzyme-labeled antibody that is not bound to the antigen in the test sample is captured by the antigen analogue that has been immobilized in advance on the purification unit, thereby obtaining a purified sample in which the free enzyme-labeled antibody has been removed from the test sample.

[0019] In the reaction unit, a substrate that is oxidized / reduced or decomposed by the enzyme of the enzyme-labeled antibody is immobilized in advance, and the enzyme of the enzyme-labeled antibody that is bound to the antigen to be detected reacts with this substrate to produce an electrode active substance.

[0020] In the detection unit, the amount of the antigen to be detected can be electrochemically measured by detecting, with the electrodes, an increase or decrease in current when the electrode active substance is oxidized in the detection unit.

[0021] Among the units that make up the antigen detection system, the purification unit captures unreacted enzyme-labeled antibodies, eliminating the need for a washing step to remove the unreacted enzyme-labeled antibodies. In addition, since the substrate substance is fixed in the reaction unit beforehand, there is no need for subsequent steps such as adding a substrate substance. As a result, the amount of antigen can be measured efficiently in a short time.

[0022] <Introduction Unit> The introduction unit can have several functions. One function is to introduce a test sample that has already been mixed with an enzyme-labeled antibody or a sample that has not yet been mixed with an enzyme-labeled antibody into the system. In this case, the purification unit, reaction unit, and detection unit installed downstream may each have independent functions, or each function may be combined in any combination.

[0023] Another function is to bind the antigen to be detected with the enzyme-labeled antibody. When the enzyme-labeled antibody is injected together with the sample, or when the enzyme-labeled antibody is pre-injected in the introduction unit, the antigen and the enzyme-labeled antibody bind in the introduction unit. Excess free labeled antibody is removed in the downstream purification unit.

[0024] Alternatively, an antigen analogue that specifically reacts with free enzyme-labeled antibodies can be immobilized in the introduction unit, and the introduction unit can have the function of simultaneously removing free enzyme-labeled antibodies that have not bound to the antigen as the antigen binds to the enzyme-labeled antibodies. In this case, the introduction unit can also have the function of a purification unit, making it possible to eliminate the downstream purification unit.

[0025] Another type is one that combines the functions of a purification unit, reaction unit, and detection unit in the introduction unit. In this case, antigens can be detected simply by dropping a sample.

[0026] The shape and material of this introduction unit are not particularly specified, and it may be made of a fibrous material such as glass fiber, etc. Appropriate selection can be made according to the intended function.

[0027] An absorbent member capable of absorbing a predetermined amount of liquid can be disposed on the end side of the support facing the introduction unit or the purification unit, thereby making it possible to stably maintain the flow of sample liquid from the purification unit toward the detection unit by utilizing capillary action.

[0028] The absorbent member used may be composed of fibers such as cellulose fiber, glass fiber, pulp, etc.; hydrophilic fibers such as nonwoven fabric and filter paper; and highly water-absorbent polymers such as polyethylene glycol, sodium polyacrylate, and polyacrylamide.

[0029] These materials can have a high water retention capacity, so that the added sample liquid can be absorbed sufficiently and at a predetermined speed. In addition, the absorption speed of the sample liquid can be increased, so that the antigen concentration can be measured quickly.

[0030] <Refining unit> The purification unit is a site where free enzyme-labeled antibodies that are not bound to antigens contained in the test sample are removed. If free enzyme-labeled antibodies that are not bound to antigens are present and come into contact with the substrate, the labeling enzyme will react with the substrate even though it is not bound to the antigen, causing noise and making it impossible to accurately measure the antigen concentration. A specified amount of antigen analog is immobilized in the purification unit beforehand, so that free enzyme-labeled antibodies in the sample liquid that is developed and supplied to the purification unit can be captured.

[0031] The purification unit may be mainly composed of a water-permeable material such as a nitrocellulose membrane, a cellulose acetate membrane, a cellulose mixed ester membrane of nitrocellulose and cellulose acetate, nylon, a nonwoven fabric, filter paper, particles, etc. Since the microstructure of the water-permeable material that serves as a liquid flow path can be changed as necessary, the speed at which the sample liquid flows through the flow path can be adjusted, and the sample can be stably measured under appropriate conditions.

[0032] Methods for immobilizing the antigen analogue include a method in which carrier particles or the like are immobilized on the water-permeable material as a carrier for the antigen analogue, and a method in which the antigen analogue is directly immobilized on the water-permeable material.

[0033] Methods for immobilization include, for example, the amine coupling method, which involves a crosslinking reaction between the carboxyl group of the carrier and the amino group in the protein, or the binding reaction between avidin and biotin. Alternatively, the protein can be directly immobilized on the support by utilizing physical adsorption, etc.

[0034] <Reaction unit> The reaction unit is a site where an enzyme-labeled antibody bound to an antigen reacts with a substrate to generate an electrode active substance. The electrode active substance generated in proportion to the amount of antigen bound to the enzyme-labeled antibody is oxidized on the electrode, and the current generated when this is detected allows the antigen to be measured with high sensitivity. The reaction unit may be present in the same space as the detection unit described below.

[0035] <Detection unit> The detection unit includes a measurement section that measures a current by applying a constant voltage between electrodes consisting of a working electrode and a counter electrode, or a working electrode, a counter electrode, and a reference electrode. The electrode is not particularly limited as long as it is made of a material, has a size and a shape that can function as an electrode, and any material can be used. Examples of the electrode include a single layer or a laminate structure of two or more layers of metals or alloys such as graphite, carbon, carbon fabric, gold, platinum, silver, copper, and aluminum, and conductive oxides such as SnO2, In2O3, WO3, and TiO2. The electrode may be formed by attaching a piece of conductive material to a substrate or burying a part of it on the substrate, or may be formed by using a printing method such as a screen printing method using a conductive paste, vapor deposition, or sputtering.

[0036] <Antigen detection method> The process of measuring electrical properties using an enzyme-labeled antibody will be described in detail. As the enzyme-labeled antibody of the present invention, both those requiring a coenzyme such as nicotinamide adenine dinucleotide (NAD) or flavin adenine dinucleotide (FAD) in the reaction with the substrate and those not requiring it can be used, and those capable of detecting the degree of reaction with the substrate by an electrical method are used. As the antibody capable of being detected by an electrical method, it is sufficient to use a substrate that is electrochemically activated when decomposed by an enzyme reaction, or an electron carrier that is reduced by a coenzyme in the presence of the coenzyme, and to measure the electrochemical response of the activated substrate or the electron carrier.

[0037] The enzyme, substrate, etc. used to bind to such an enzyme-labeled antibody and measure the electrical properties are appropriately selected depending on the antigen and enzyme-labeled antibody, and examples include a combination of alkaline phosphatase as the enzyme and p-aminophenyl phosphate as the substrate, a combination of horseradish peroxidase as the enzyme, hydrogen peroxide as the substrate, and a mixed solution of ferrocene and methanol, glucose oxidase or lactate oxidase as the enzyme, glucose or lactate as the substrate, etc. In the present invention, FAD or NAD may be used as the coenzyme, and ferricyanide or vitamin K may be used as the electron carrier.

[0038] When detecting immunoglobulin A, which is one of the preferred embodiments of the present invention, p-aminophenylphosphate (hereinafter abbreviated as "pAPP") can be used as a substrate that binds to the enzyme-labeled antibody of the present invention. This pAPP is decomposed by an enzyme (alkaline phosphatase) to become p-aminophenol (pAP), which is then oxidized to p-quinoneimine (pQI). By detecting the electrochemical response during this process, the presence or absence of an antigen-antibody reaction can be detected by an electrical method as a change in current or voltage.

[0039] When performing detection by an electrical method, various amperometry and voltammetry are preferred, although there is no particular limitation. Amperometry is a method for measuring and analyzing the current flowing through an electrode with respect to a potential step versus time, and is a method that is widely used for quantitative analysis of electrochemically active species in a sample solution. Voltammetry is a method that is widely used as a method for measuring the response current when the electrode potential is changed. There are cyclic voltammetry, which changes the electrode potential linearly, and pulse voltammetry, which changes it by a pulse. The response current value obtained depends on the concentration of the electrochemically active species in the sample solution. In the present invention, the electrochemical response of the activated substrate generated in the solution is measured using such known electrochemical methods and means, and the original antigen is qualitatively and / or quantitatively analyzed.

[0040] <Sample> The sample in the present invention refers to a test object in a general clinical test, etc. Specific samples include blood (whole blood, plasma, serum), lymph, saliva, urine, feces, sweat, mucus, tears, cerebrospinal fluid, nasal discharge, cervical or vaginal secretions, semen, pleural fluid, amniotic fluid, peritoneal fluid, middle ear fluid, synovial fluid, gastric aspirate, biological fluids such as tissue / cell extracts and homogenates, as well as solutions such as food, soil, and plant extracts and homogenates, river water, hot spring water, drinking water, polluted water, etc. Almost all liquid samples are used. In the present invention, since only one type of antibody is required for the detection object, not only a substance with a large molecular weight to which multiple monoclonal antibodies can bind, but also a low molecular weight substance (hapten) to which only one monoclonal antibody can bind can be the detection object.

[0041] <Antigen analogues> The antigen analog used in the antigen detection system of the present invention is not particularly limited as long as it specifically binds to the labeled antibody through an antigen-antibody reaction to form a conjugate. For example, compounds such as cadmium and dioxin, various biomolecules such as viral proteins and steroids, low molecular weight antibodies, aptamers, etc. can be used. In the present invention, the antigen analog is immobilized on the purification unit of the antigen detection system. Therefore, it is preferable that the antigen analog has high storage stability. Specific examples include stress markers such as immunoglobulin A / IgA and chromogranin A / CgA, and inflammatory markers such as anti-inflammatory cytokines.

[0042] <How to prepare enzyme-labeled antibodies> For enzyme-labeled antibodies, commercially available enzyme-labeled antibody reagents, which are pre-labeled with an enzyme that reacts with the substrate to be used, such as peroxidase or alkaline phosphatase, can be used. Alternatively, enzyme-labeled antibodies can be prepared by oneself using commercially available enzyme labeling kits. In this case, enzymes such as alkaline phosphatase are pre-activated with succinimidyl ester groups or maleimide groups, and a kit can be used that allows easy enzyme labeling of the amino or thiol groups of the antibody molecule to be labeled by simply mixing the enzyme with the target antibody molecule. For example, in the case of peroxidase, Peroxidase Labeling Kit-NH2 (product number LK11) or Peroxidase Labeling Kit-SH (product number LK09) manufactured by Dojindo Laboratories can be used, and in the case of alkaline phosphatase, Alkaline Phosphatase Labeling Kit-NH2 (product number LK12) or Alkaline Phosphatase Labeling Kit-SH (product number LK13) can be used to easily label the enzyme.

[0043] <Form of antigen detection system> The antigen detection system of the present invention may take the form of, for example, a test strip, a biomarker detection chip, and the like.

[0044] <Support for antigen detection system> The material of the support may be any resin that is lightweight and easy to dispose of, such as acrylic, polycarbonate, polypropylene, or polystyrene.

[0045] <Antigen detection kit> The antigen detection kit includes at least an introduction unit containing an enzyme-labeled antibody solution or an enzyme-labeled antibody, and an antigen detection system including a purification unit on which an antigen analog is immobilized, a reaction unit on which a substrate is immobilized, a detection unit having an electrode, and an absorption unit. The antigen detection system may further include a support on which the antigen detection system is immobilized. EXAMPLES

[0046] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Note that, unless otherwise specified, units are based on mass.

[0047] [Reagents and materials] Membrane: Immunochromatographic nitrocellulose membrane (Immunopore RP), (GE Healthcare, code number 78356403) Antigen analog binding filter membrane: Cellulose mixed ester type membrane filter (Advantec Toyo Co., Ltd., product number A500A090C) Absorbent pad: Immunochromatographic absorbent pad (CF4), (GE Healthcare, code number 8114-2250) Alkaline phosphatase-labeled anti-immunoglobulin A antibody: Goat Anti-Human IgA alpha chain (Alkaline Phosphatase) preadsorbed, (Abcam, ab98551) Immunoglobulin A antigen: Native Human IgA protein (Abcam, ab91025) p-Aminophenylphosphate (pAPP): 4-Aminophenylphosphate Monosodium, (CAS number: 52331-30-3), (LKT Laboratories, product number A5030) Phosphate buffered saline (PBS): 137mmol / l NaCl, 8.1mmol / l Na2HPO4, 2.68mmol / l KCl, 1.47mmol / l KH2PO4 pH 7.4 Tris-buffered saline (TBS): 150mmol / l NaCl, 20mmol / l Tris, adjusted to pH 7.4 and pH 8.0 Bovine serum albumin (BSA): (CAS number: 9048-46-8), (Sigma-Aldrich, Catalog number: A4503) Blocking reagent: SuperBlock (TBS) Blocking Buffer, (Thermo Fisher Scientific KK, product number 37535) Surfactant: Polyoxyethylene (20) sorbitan monolaurate, Tween (TM) Equivalent to 20, (CAS number: 9005-64-5), (Wako Pure Chemical Industries, Ltd., product code number: 167-11515) Potassium chloride (KCl): (CAS number 7447-40-7), (Wako Pure Chemical Industries, Ltd., product number 169-03542) Working electrode: Polyethylene naphthalate (PEN) film, Teijin DuPont Films' PEN film Q51-A4-188 μm substrate with 50 nm thick gold vacuum-deposited on it (Figure 3). Reference electrode: Silver-silver chloride ink for reference electrodes (manufactured by BAS, catalog number 011464). Silver-silver chloride ink for reference electrodes was dropped onto the working electrode and dried in a dryer at 60°C for 2 hours (Figure 3). Electrode holder: Polydimethylsiloxane, SILPOT, a silicone potting material manufactured by Dow Corning Toray TM Silicone resin sheet using 184. Resin sheet is made by SILPOT TM The base resin and hardener of 184 were mixed in a ratio of 20:1, and after degassing with a vacuum pump while heating to 75℃, the resin liquid was poured into a 5mm x 10mm x 1mm mold, spread to a uniform thickness, and left to stand at 75℃ for 1 hour.

[0048] [Experimental equipment] Measurement equipment: ALS / CH Instruments electrochemical analyzer ALS704E, manufactured by BAS.

[0049] [Electrochemical detection method for biological materials] The electrochemical detection method for biological substances in the examples included the following steps: blocking, holding the substrate, immobilizing the antigen analog, assembling the detection device, contacting the antigen to be detected with the enzyme-labeled antibody, and measuring the electrical properties of the membrane.

[0050] 1.Blocking step A GE Healthcare immunochromatographic membrane (Immunopore RP) was cut into a length of 50 mm and a width of 5 mm. To suppress non-specific adsorption, a membrane blocking buffer was prepared by adding 0.5% of the surfactant polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20) to Thermo Fisher Scientific's SuperBlock (TBS) Blocking Buffer solution (product number 37535). The cut immunochromatographic membrane was immersed in this blocking buffer and subjected to blocking treatment by immersing it in the blocking buffer for 60 minutes while shaking with a plate mixer (Taitec, tabletop small shaker, Mild Mixer PR-36). A washing solution of 20 mM TBS-T (pH 8.0) containing 0.5% of the surfactant polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20) was prepared. The cut immunochromatographic membrane was immersed in this washing solution and washed for 30 minutes while being shaken with a plate mixer (Taitec Corporation, tabletop small shaker, Mild Mixer PR-36). The membrane was removed from the washing solution and left to stand overnight in a refrigerator at 4°C.

[0051] 2. Substrate retention process A 100 mM pAPP solution was prepared by dissolving p-aminophenyl phosphate (pAPP), a substrate for alkaline phosphatase, in purified water. 2 μL of 100 mM pAPP solution was dropped onto a membrane that had been previously blocked, 20 mm from the end, and the membrane was left to stand at 37° C. for 2 hours to retain the substrate. The membrane retaining the substrate was then stored at 4° C. until use. The substrate solution may be dropped either before or after the membrane is immersed in the blocking solution, but it is preferable to drop the substrate after immersion, since a higher electrochemical signal can be obtained. After dropping the substrate solution, the membrane may be stored at a low temperature, but the dropped substrate must be thoroughly dried.

[0052] 3. Immobilizing the antigen analogue In order to capture free alkaline phosphatase-labeled anti-immunoglobulin A antibodies that do not react with the antigen to be detected, immunoglobulin A antigen was immobilized in advance as an antigen analog on a cut membrane filter or on the immunochromatographic membrane of the flow path by the following method. In the method of immobilizing the antigen on a membrane filter, a cellulose mixed ester type membrane filter (Advantec Toyo Co., Ltd., product number A500A090C) was used as the membrane filter, which was cut to 5 mm x 7 mm, and 2 μL of immunoglobulin A antigen (Native Human IgA protein, (Abcam, ab91025) adjusted to 1 mg / mL with PBS containing 100 ng / mL BSA was dropped onto the membrane filter at 4 locations for a total of 8 μL. In the examples, the membrane filter to which the immunoglobulin A antigen was bound was not subjected to a blocking treatment to suppress non-specific adsorption, but if it is desired to further suppress non-specific adsorption, a blocking treatment may be performed using an appropriate blocking agent. In addition to the cellulose mixed ester type, a PVDF membrane that has a pore size suitable for the passage of liquid and is capable of binding protein molecules, or a column packed with bead particles capable of binding protein molecules, etc., can also be used as the membrane filter to which the immunoglobulin A antigen is bound.

[0053] In the method of immobilizing the antigen analogue on the immunochromatographic membrane of the flow channel, a GE Healthcare immunochromatographic membrane (Immunopore RP) was cut to a length of 50 mm and a width of 5 mm, and 2 μL of immunoglobulin A antigen (Native Human IgA protein, Abcam, ab91025) adjusted to 1 mg / mL in PBS containing 100 ng / mL BSA was dropped onto the membrane at a position 13 mm from the end of the membrane on the sample injection port side. To suppress non-specific adsorption, a membrane blocking buffer was prepared by adding 0.5% of the surfactant polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20) to Thermo Fisher Scientific SuperBlock (TBS) Blocking Buffer solution (product number 37535). The immunochromatographic membrane with the antigen analogue dropped onto it was immersed in this blocking buffer and mixed with a plate mixer (Taitec, small tabletop shaker). The membrane was immersed in the blocking buffer for 60 minutes while being shaken with a mild mixer (PR-36). A washing solution of 20 mM TBS-T (pH 8.0) containing 0.5% of the surfactant polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20) was prepared. The cut immunochromatographic membrane was immersed in this washing solution and washed for 30 minutes while being shaken with a plate mixer (Taitec, small tabletop shaker, mild mixer PR-36). The membrane was removed from the washing solution and left to stand overnight in a refrigerator at 4°C. Then, following the above-mentioned "substrate retention process", p-aminophenyl phosphate (pAPP), a substrate for alkaline phosphatase, was dissolved in purified water to prepare a 100 mM pAPP solution. 100 mM pAPP was added to the membrane 23 mm from the end of the membrane that had been previously blocked. 1.25 μL of pAPP solution was dropped, and 5 minutes later, 1.25 μL was further dropped, and the membrane was left to stand at 37° C. for 2 hours to retain the substrate. Thereafter, the membrane retaining the substrate was stored at 4° C. until use.

[0054] 4. Assembling the detection device (1) Preparation of gold electrode for detection The gold detection electrode with the electrode wiring shown in Figure 3 was prepared by depositing gold to a thickness of 50 nm on a polyethylene naphthalate (PEN) film (Teijin DuPont Films, PEN film Q51-A4- 188 μm) substrate using a vacuum deposition device. Furthermore, silver-silver chloride ink for reference electrodes (manufactured by BAS, catalog number 011464) was applied to the central part of the part that contacts the flow channel of the immunochromatography membrane shown in Figure 3 on the middle wire of the three wires of the electrode in the position shown in Figure 3, and dried at 60°C for 2 hours.

[0055] (2) Electrode holder The silicone resin used was polydimethylsiloxane (SILPOT® 184, a silicone potting material manufactured by Dow Corning Toray Co., Ltd.). The base agent and curing agent of SILPOT® 184 were mixed at a ratio of 20:1, and the mixture was heated to 75°C and degassed using a vacuum pump. The prepared resin liquid was then poured into a mold measuring 5 mm x 10 mm x 1 mm and spread to a uniform thickness. The mixture was then left to stand for 1 hour at 75°C, after which it was removed from the mold to produce a silicone resin sheet.

[0056] (3) Preparation of absorbent pads An immunochromatographic absorbent pad (CF4) (GE Healthcare, code number 8114-2250) was cut to 14.7 mm x 17 mm.

[0057] (4) Assembling the detection device The detection device was constructed as shown in Figures 1 and 2, with a previously prepared substrate-holding immunochromatographic membrane 2, an antigen analog binding filter 7, a detection gold electrode 3 coated with reference electrode ink, an absorbent pad 4, and a silicone resin sheet (5 mm x 10 mm x 1 mm) 8 to ensure contact between the substrate-holding immunochromatographic membrane 2 and the detection gold electrode 3 attached to the back of the electrodes on top of an acrylic immunochromatographic housing 6. The antigen analogue binding filter was placed in contact with the immunochromatographic membrane near the sample injection port 1 of the detection device shown in Example 1. In Example 2, when an immunochromatographic membrane on which an antigen analogue is dropped at a position 13 mm from the end is used, the antigen analogue binding filter 7 becomes unnecessary. In addition, a filter holder capable of mounting a filter membrane can be used to place the filter in contact with the sample injection port 1, or it can be used as a tool for a sample pretreatment step.

[0058] 5. A step of contacting the antigen to be detected with the enzyme-labeled antibody The test solution for each Example was prepared by mixing 11 μL of immunoglobulin A antigen solution adjusted to a test concentration with TBS (pH 8.0) containing 0.1% BSA and 0.05% polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20), 11 μL of alkaline phosphatase-labeled anti-immunoglobulin A antibody solution adjusted to 10 μg / mL with TBS (pH 8.0) containing 0.1% BSA and 0.05% polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20), and 22 μL of a sample preparation solution previously mixed with 425 μL of TBS (pH 8.0), 25 μL of 1M KCl, and 50 μL of purified water in a microtube.

[0059] 6. Measuring the electrical properties of the membrane The terminal of the detection gold electrode 3 shown in FIG. 1 and FIG. 2 was connected to the measurement device, ALS / CH Instruments electrochemical analyzer ALS704E (BAS Corporation), via a connector cable.

[0060] 40 μL of test solution containing immunoglobulin A antigen was dropped from the sample injection port 1 of this detection device. After the injection of the test solution, the electrochemically generated current value was measured using an electrochemical analyzer by the chronoamperometry technique (it) under the following measurement conditions, starting from the point when 20 seconds had elapsed after the injection of the test solution. Measurement parameters of the Amperometry IT technique: Initial potential (V)=0.15 Sample Interval (Sec) = 0.1 Measurement time (Sec) =2e+3 Quiet time (Sec) =0 Sensitivity (A / V)=1e-6

[0061] When the test solution contained immunoglobulin A antigen, which was the target antigen, the enzyme activity of alkaline phosphatase in the enzyme-labeled antibody bound to the antigen caused the substrate p-aminophenyl phosphate to be oxidized to p-quinoneimine via p-aminophenol, and the current generated was quantitatively detected. Furthermore, the current value for each elapsed measurement time was calculated as the integrated value (integral command processing) using the following formula, and the amount of charge (Coulomb·C) detected in a certain period of time was calculated. To obtain a more accurate concentration of the target antigen, a calibration curve of antigen concentration and charge amount was created, allowing for an estimate. Current (A) x time (S) = charge (C)...Equation 1 The charge amounts obtained for each test concentration of immunoglobulin A antigen contained in the test solution are shown in FIG.

[0062] [Example 1] In the electrochemical detection method for biological substances in the present embodiment described above, an antigen-binding filter in which immunoglobulin A antigen had been immobilized in advance on a cellulose mixed ester membrane was used to capture unreacted alkaline phosphatase-labeled anti-immunoglobulin A antibody. In addition, 11 μL of immunoglobulin A antigen solution was prepared with TBS (pH 8.0) containing 0.1% BSA and 0.05% polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20) to test concentrations of 0, 1, 10, and 100 μg / mL, 11 μL of alkaline phosphatase-labeled anti-immunoglobulin A antibody solution was prepared with TBS (pH 8.0) containing 0.1% BSA and 0.05% polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20) to test concentrations of 10 μg / mL, and 425 μL of TBS (pH 8.0) and 1M KCl A test solution was prepared by mixing 25 μL of immunoglobulin A and 22 μL of a solution for sample preparation, which was a mixture of 50 μL of purified water and 25 μL of IgG, in a microtube. The current value electrochemically generated was measured with an electrochemical analyzer using this detection device, and the integrated value calculated by formula 1 / amount of charge (Coulomb·C) detected in a certain time period is shown in Figure 4. As shown in Figure 4, the electrochemical detection method of the present invention is capable of detecting concentrations from 0.1 μg / mL to 100 μg / mL, and the presence or absence of immunoglobulin A antigen could be confirmed from the detected charge value (Coulomb·C), and its concentration could also be calculated.

[0063] [Example 2] In the electrochemical detection method of the present embodiment, in order to capture unreacted alkaline phosphatase-labeled anti-immunoglobulin A antibodies, immunoglobulin A antigens were dropped onto a cut immunochromatographic membrane at a position 13 mm from the end of the membrane on the sample injection port side, and the immunochromatographic membrane was blocked with Thermo Fisher Scientific's Super Block solution containing 0.5% of the surfactant polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20), and further p-aminophenyl phosphate (pAPP), a substrate for alkaline phosphatase, was dropped onto the membrane at a position 23 mm from the end. The immunochromatographic membrane used was the same as that used in Example 1. The immunoglobulin A antigens to be detected, 10 μg / mL of anti-immunoglobulin A antibodies labeled with alkaline phosphatase, and phosphate buffered saline were mixed to prepare a test solution to be tested. As shown in Figure 5, the presence or absence of immunoglobulin A antigen could be confirmed from the charge (Coulomb·C) at anti-immunoglobulin A antigen concentrations of 0, 1, and 10 μg / mL, and the concentration could also be calculated.

[0064] [Comparative Example 1] In addition to the reagents and materials used in Examples 1 and 2, a nitrocellulose membrane for immunochromatography (HF120) manufactured by Millipore and a primary antibody, anti-immunoglobulin A antibody: Anti-Human IgA antibody [3B7] (manufactured by Abcam, ab7400), were used as the antigen capture antibody immobilized on the immunochromatography membrane.

[0065] Nitrocellulose membrane for immunochromatography (HF120) manufactured by Millipore was cut to a length of 50 mm and a width of 5 mm. 2 μl of primary antibody solution, which was prepared by diluting anti-immunoglobulin A antibody (Anti-Human IgA antibody [3B7] (manufactured by Abcam, ab7400) to 1 mg / ml with PBS, was dropped onto the immunochromatography membrane at a position 20 mm from the end, and the membrane was dried at 37°C for 2 hours.

[0066] The immunochromatographic membrane with the immobilized primary antibody was immersed in SuperBlock T20 (TBS) Blocking Buffer (product number 37536) from Thermo Fisher Scientific, and left to soak for 15 minutes while shaking with a plate mixer. Using a pipetting reservoir, the blocked immunochromatographic membrane was immersed in a washing solution of TBS-T (pH 7.4) containing 0.05% polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20), and washed for 30 minutes while shaking with a plate mixer. The membrane was removed from the washing solution and left to stand overnight in a refrigerator at 4°C.

[0067] The prepared immunochromatographic membrane, absorbent pad, gold electrode for detection, and a silicone resin sheet for ensuring contact between the immunochromatographic membrane and the detection electrode were each set in an immunochromatographic housing to assemble the detector.

[0068] 10 μL of immunoglobulin A antigen solution, adjusted to test concentrations of 0, 1, and 5 μg / mL with TBS-T containing 0.1% BSA and 0.05% polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20), and 10 μL of alkaline phosphatase-labeled anti-immunoglobulin A antibody: Goat Anti-Human IgA alpha chain (Alkaline Phosphatase) preadsorbed (Abcam, ab98551), adjusted to 10 μg / mL with TBS-T containing 0.1% BSA and 0.05% polyoxyethylene (20) sorbitan monolaurate (equivalent to Tween 20), were successively dropped from the sample injection port of the detector.

[0069] After the liquid in the sample injection port had run out, 50 μL of 5 mM pAPP substrate solution prepared with a mixture of 20 mM TBS (pH 8.0) and 50 mM KCl was added. 10 seconds after the addition of the substrate solution, electrochemical measurement was started and the current value was detected in it amperometry mode. The difference in the current value after 50 seconds and 200 seconds of measurement was taken as the detected current value. Measurement parameters in amperometry (it) mode: Initial potential (V)=0.15 Sample Interval (Sec) = 0.1 Measurement time (Sec) = 400 Quiet time (Sec) =0 Measurement scale = 1 Sensitivity (A / V)=1e-6

[0070] As a result, in the comparative example in which the washing step of not dropping washing solution after adding the enzyme-labeled antibody reagent was omitted, free alkaline phosphatase-labeled anti-immunoglobulin A antibodies that were not bound to the antigen to be detected remained on the immunochromatographic membrane near the electrode. The alkaline phosphatase labeled to the free antibodies increased the electrical noise, making it impossible to detect an electrical signal that depended on the concentration of the immunoglobulin A antigen to be detected. [Industrial Applicability]

[0071] The present invention relates to a method for evaluating an antigen-antibody reaction that can be evaluated in a short time and provides quantitative measurement results, and relates to an evaluation method that can detect, for example, immunoglobulin A, a stress marker, and contributes to the provision of an antigen detection system that can be used at the site of POCT, etc. [Explanation of symbols]

[0072] 1...Sample injection port, 2...Substrate-holding immunochromatographic membrane, 3...Gold electrode for detection, 4...Water-absorbing pad, 5...Housing (upper side), 6...Housing (lower side), 7...Antigen analog binding filter, 8...Electrode holder, 9...Position of silver-silver chloride ink drop for reference electrode

Claims

1. An antigen detection system using an enzyme-labeled antibody that reacts with an antigen, A transfer unit which may include an enzyme-labeled antibody; a purification unit on which an antigen analogue that reacts with the enzyme-labeled antibody is immobilized; a reaction unit having a substrate immobilized thereon that reacts with the enzyme of the enzyme-labeled antibody; A detection unit for detecting an increase or decrease in an electrical signal, the antigen analog and the substrate are immobilized on a single support; The detection unit has an electrode having a working electrode and a counter electrode.

2. The antigen detection system according to claim 1 , wherein the reaction unit and the detection unit are present on the same flow path.

3. 3. The antigen detection system according to claim 1, wherein the sample liquid is allowed to move through the introduction unit, the purification unit, the reaction unit, and the detection unit in this order.

4. 4. The antigen detection system according to claim 1, wherein the enzyme of the enzyme-labeled antibody is at least one selected from the group consisting of hydrolases and oxidoreductases.

5. The antigen detection system according to any one of claims 1 to 4, wherein the support for immobilizing the antigen analog in the purification unit is at least one selected from the group consisting of a membrane filter, an immunochromatographic membrane, a nonwoven fabric, a filter paper, a mesh, and a carrier particle.

6. 6. An antigen detection kit comprising the antigen detection system according to any one of claims 1 to 5 immobilized on a support, and an enzyme-labeled antibody.

7. An antigen detection method using the antigen detection system according to any one of claims 1 to 6, (1) a first step of contacting a sample containing an antigen with an enzyme-labeled antibody to obtain a test sample; (2) a second step of contacting the test sample obtained in the first step with a purification unit to capture unreacted enzyme-labeled antibodies in the purification unit to obtain a purified sample; (3) a third step of contacting the purified sample with the reaction unit to react the enzyme-labeled antibody with the substrate; and (4) A fourth step of detecting an electric signal generated by contacting the reaction product of the third step with a detection unit. A method for detecting an antigen, comprising:

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