Immunochromatographic test strips

Abstract

The problem to be solved by the present invention is to provide a test piece that can avoid the so-called bleaching phenomenon in the immunochromatographic test piece, so as to achieve better detection and visibility. Another problem to be solved by the present invention is to provide an immunochromatographic test piece that can prevent the variation of the detection value on the test line. The present invention solves the aforementioned problems by an immunochromatographic test piece that at least includes a porous membrane and has a sample application area, a development area and a detection area fixed with a specific binding substance, wherein the detection area is fixed on the surface of the porous membrane, and wherein an opaque substrate with a thickness of more than 30 μm and less than 200 μm is provided on the back of the porous membrane.

Description

Technical Field

[0001] The present invention relates to an immunochromatographic test piece and an immunochromatographic detection method using the test piece. Background Art

[0002] With the spread of POCT (point of care testing) for near-patient treatment, lateral flow immunochromatographic detection methods using test pieces made of nitrocellulose membranes or the like have been widely used as simple immunoassays using antigen-antibody reactions.

[0003] The test strip based on immunochromatography (referred to herein as immunochromatography test strip) is usually composed of a sample application area, an expansion area and a detection area located on a porous membrane. The membrane is configured so that the detection antibody targeting a specific analyte binds to the sample and can reach the detection area along the expansion area downward. The antibody responsible for analyte detection is fixed at a certain position on the expansion area called the detection area.

[0004] When a sample is dripped onto the sample application area and the sample contains an analyte, the analyte specifically binds to the labeled antibody to form a complex. The complex is developed in the downstream direction of the development area and binds to the fixed antibody to be detected in the detection area. Therefore, by detecting the sandwich complex of the labeled antibody, the analyte and the fixed antibody in the detection area, the analyte can be qualitatively or quantitatively analyzed.

[0005] The method for detecting the complex of the analyte and the conjugate in the detection area of ​​the immunochromatographic test strip having the above-mentioned structure includes a method of measuring the intensity of the reflected light from the label to calculate the absorbance (reflected absorbance). The reflected absorbance is calculated by using the ratio of the reflected light intensity measured on the detection line of the analyte to the two adjacent areas on both sides of the detection area. If this method is used, the measurement waveform may be disturbed due to the non-specific increase in the reflected light intensity near the upstream side or downstream side of the detection area of ​​the detection complex. In this case, when the concentration of the analyte is low, the interference of the measurement waveform cannot be ignored and the baseline cannot be subtracted, and the specific signal (peak) cannot be accurately detected, which may cause the measurement itself to be unable to be performed.

[0006] Since the interference frequency in the measurement waveform and the degree of disturbance in the measurement waveform are not constant, the repeatability of each measurement is reduced, and correction to an appropriate measurement value (in other words, confirmation of the measurement waveform) must be made in each measurement.

[0007] In conventional immunochromatographic detection methods using white membranes, disturbances in the measurement waveform can be visually observed as a phenomenon in which a specific location on the membrane appears relatively whiter than the surrounding color, as if the location is bleached (so-called bleaching phenomenon; hereinafter also referred to simply as bleaching). This prevents visual confirmation of the color produced by the marker and may result in a decrease in the accuracy of the test result.

[0008] Although the cause of this bleaching is not clear, it is presumed to be caused by differences in the production volume of the test pieces, the storage conditions of the test pieces, and the production conditions (eg, pretreatment conditions) of the test pieces.

[0009] As a method for eliminating this bleaching phenomenon, the following documents are known.

[0010] Patent Document 1 discloses an assay method using a sample diluent to which methanol is added in order to prevent bleaching that is often observed when immunochromatographic test strips are stored for a long period of time.

[0011] Patent Document 2 discloses a method for preventing bleaching and interference with a measurement waveform by treating an antibody-immobilized membrane of an immunochromatographic test piece with a specific surfactant such as n-octyl-β-D-glucoside.

[0012] Citation List

[0013] Patent Literature

[0014] Patent document 1: WO 2015 / 080286

[0015] Patent document 2: WO 2010 / 001598 Summary of the invention

[0016] Technical issues

[0017] The problem to be solved by the present invention is to provide a test strip that can avoid the so-called bleaching phenomenon in the immunochromatographic test strip by a method completely different from the prior art, and perform detection with better visibility. Another problem to be solved by the present invention is to provide an immunochromatographic test strip that can prevent the detection value on the test line from varying.

[0018] Solutions to the problem

[0019] The inventors attempted to change various elements of the immunochromatographic test piece to solve this problem, and surprisingly found that, in an immunochromatographic test piece including a porous membrane, by providing an opaque substrate on the back of the porous membrane having a specific binding substance such as an antibody fixed on the surface, bleaching near the test line can be avoided and variation in the detection value can be prevented, thereby completing the present invention. Specifically, the present invention has the following configuration.

[0020] (1) An immunochromatographic test piece comprising at least a porous membrane, the immunochromatographic test piece comprising a sample application region, a development region and a detection region, wherein the detection region is fixed on the surface of the porous membrane, and wherein an opaque substrate is provided on the back side of the porous membrane.

[0021] (2) The immunochromatographic test piece according to (1), wherein the opaque substrate is a white substrate.

[0022] (3) The immunochromatographic test piece according to (1) or (2), wherein the opaque substrate has a thickness of 30 μm or more and 200 μm or less.

[0023] (4) The immunochromatographic test piece according to any one of (1) to (3), wherein the opaque substrate is provided for lining the porous membrane, and wherein a liner sheet is provided below the opaque substrate.

[0024] (5) The immunochromatographic test piece according to any one of (1) to (4), further comprising a sample pad serving as a sample application area, and a conjugate pad containing a specific binding substance labeled with colloidal gold or colored latex.

[0025] (6) The immunochromatographic test piece according to any one of (1) to (5), wherein the specific binding substance is an antibody.

[0026] (7) The immunochromatographic test piece according to any one of (1) to (6), wherein the immunochromatographic test piece is a test piece for detection by optical means.

[0027] (8) An immunochromatographic test kit comprising: the immunochromatographic test piece according to any one of (1) to (7).

[0028] (9) An immunochromatographic detection method, comprising: using the following test strip:

[0029] An immunochromatographic test piece includes a porous membrane, the porous membrane includes at least a detection area, and the porous membrane has an opaque substrate disposed on the back side of the porous membrane.

[0030] (10) The immunochromatographic detection method according to (9), wherein the opaque substrate is a white substrate.

[0031] (11) The immunochromatographic detection method according to (9) or (10), wherein the thickness of the opaque substrate is 30 μm or more and 200 μm or less.

[0032] (12) The immunochromatographic detection method according to any one of (9) to (11), wherein the opaque substrate is provided for lining the porous membrane, and wherein a liner is provided below the opaque substrate.

[0033] (13) The immunochromatographic detection method according to any one of (9) to (12), wherein the test piece further comprises a sample pad serving as a sample application area, and a conjugate pad comprising a specific binding substance labeled with colloidal gold or colored latex.

[0034] (14) The immunochromatographic detection method according to any one of (9) to (13), wherein the specific binding substance is an antibody.

[0035] (15) The immunochromatographic detection method according to any one of (9) to (14), comprising the step of detecting an optical parameter derived from the labeling substance by optical means.

[0036] (16) The immunochromatographic detection method according to any one of (9) to (15), comprising the step of determining the color development derived from the labeling substance by visual observation.

[0037] (17) A method for reducing the variation of measured values ​​in an immunochromatographic detection method, the method comprising: using the following test strip:

[0038] An immunochromatographic test piece comprising at least a porous membrane, wherein the immunochromatographic test piece comprises a sample application area, a development area and a detection area, wherein the detection area is fixed on the surface of the porous membrane, and wherein

[0039] An opaque substrate is disposed on the back side of the porous membrane.

[0040] (18) The method for reducing variation according to (17), wherein the opaque substrate is a white substrate.

[0041] (19) The method for reducing variation according to (17) or (18), wherein the thickness of the opaque substrate is 30 μm or more and 200 μm or less.

[0042] (20) The method for reducing variation according to any one of (17) to (19), wherein the opaque substrate is a member provided for inner lining of the porous membrane, and wherein a lining sheet is provided below the opaque substrate.

[0043] (21) A method for reducing bleaching of a test strip in an immunochromatographic detection method using a test strip, the method comprising: using the following test strip:

[0044] An immunochromatographic test piece comprising at least a porous membrane, wherein the immunochromatographic test piece comprises a sample application area, a development area and a detection area, wherein the detection area is fixed on the surface of the porous membrane, and wherein an opaque substrate is provided on the back side of the porous membrane.

[0045] (22) The method for reducing bleaching of a test piece according to (21), wherein the opaque substrate is a white substrate.

[0046] (23) The method for reducing bleaching of a test piece according to (21) or (22), wherein the thickness of the opaque substrate is 30 μm or more and 200 μm or less.

[0047] (24) The method for reducing bleaching of a test piece according to any one of (21) to (23), wherein the opaque substrate is provided for lining the porous membrane, and wherein a lining sheet is provided below the opaque substrate.

[0048] Advantageous Effects of the Invention

[0049] The present invention can provide an immunochromatographic test piece which can avoid bleaching near a test line and thus has excellent visibility at the time of visual inspection and results in less variation in values ​​measured by optical means. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] [ Figure 1 ] Figure 1 It is a graph showing changes in measured values ​​when immunochromatography measurement is performed using an immunochromatography test piece using a porous membrane, wherein the liner is made of a transparent substrate (Comparative Example) or a white substrate (Present Invention). (Test Example 2, analyte at the middle concentration side)

[0051] [ Figure 2 ] Figure 2 It is a graph showing changes in measured values ​​when immunochromatography measurement is performed using an immunochromatography test piece using a porous membrane, wherein the liner is made of a transparent substrate (Comparative Example) or a white substrate (Present Invention). (Test Example 3, analyte on the low concentration side)

[0052] [ Figure 3 ] Figure 3 It is a graph showing changes in measured values ​​when immunochromatography measurement is performed using an immunochromatography test piece using a porous membrane, wherein the liner is made of a transparent substrate (Comparative Example) or a white substrate (Present Invention). (Test Example 4, analyte on the high concentration side)

[0053] [ Figure 4 ] Figure 4 These are perspective views showing the immunochromatographic test piece of the present invention. A shows the entire immunochromatographic test piece, and B shows the porous membrane.

[0054] [ Figure 5 ] Figure 5 It is a top view of the immunochromatographic detection device of the present invention.

[0055] [ Figure 6 ] Figure 6Display when cutting along the dotted line Figure 5 The device shown is a cut cross section. DETAILED DESCRIPTION

[0056] (Immunochromatographic test strip)

[0057] The immunochromatographic test piece of the present invention comprises at least a "sample application area", a "development area" and a "detection area" within the porous membrane. The labeled specific binding substance bound to the analyte is retained in a manner that allows the sample to be eluted and then flow through the development area and reach the detection site. In addition, the specific binding substance used for detection is fixed to a specific portion of the development area constituting the detection area.

[0058] An example of a test piece that embodies these elements is a test piece that includes a sample pad used as a sample application area, a conjugate pad that holds an elutable labeled specific binding substance that binds to an analyte as part of a development area, and a porous membrane that can fix the specific binding substance for detection to a specific site on the membrane and serves as a development area and a detection area. Specifically, a typical test piece for immunochromatography of the present invention has the following structure:

[0059] (1) Sample pad for loading samples;

[0060] (2) a conjugate pad disposed downstream of the sample pad and holding the conjugate sensitized by the first specific binding substance on the colloidal gold surface in an elutable manner;

[0061] (3) A porous membrane disposed downstream of the conjugate pad and having a detection area on the surface, wherein the second specific binding substance binds to a complex formed by the conjugate and the analyte, thereby immobilizing the analyte, the porous membrane having an opaque substrate disposed on the back side.

[0062] The sample pad, conjugate pad and porous membrane may each constitute a separate carrier, or two of them may constitute one carrier, and any form may be used as long as the sample pad, conjugate pad and porous membrane are arranged in this order from upstream to downstream.

[0063] In addition to the above-mentioned constitution, the immunochromatographic test piece may have one or more absorption pads and a third pad further disposed and mounted thereon.

[0064] The test piece is usually arranged on a solid support, such as a plastic adhesive sheet (hereinafter also referred to as a liner).

[0065] In order to increase the mechanical strength of the porous membrane to which the specific binding substance is immobilized and prevent evaporation (drying) of water during measurement, a polyester film or the like may be laminated on the test piece.

[0066] The detection method using the immunochromatographic test piece of the present invention is performed as follows.

[0067] When the sample is dripped onto the sample application area and the sample contains analyte, the analyte specifically binds to the specific binding material of the mark, thereby forming a complex. The complex is expanded in the expansion area in the downstream direction and is combined with the specific binding material fixed in the detection area. In the detection area, a sandwich complex of the specific binding material of the mark, the analyte and the immobilized specific binding material is formed, and therefore, the analyte can be qualitatively or quantitatively analyzed by detecting the complex. The example of detection includes a method for determining color development by visual observation and a method for detecting optical parameters derived from the mark by optical means. When using optical means, detection can be performed manually or automatically, and continuous measurement can also be performed.

[0068] (Sample Pad)

[0069] The sample pad used in the present invention is a site serving as a sample application area for receiving a sample, and any materials and forms may be used as long as those materials in a state of being molded into a pad can absorb a liquid sample and allow components of the liquid and analyte to pass therethrough.

[0070] The sample pad of the present invention may be pre-treated to increase permeability.

[0071] When whole blood is measured, a hemagglutinating agent may be added. In this case, the agent may be contained in at least a portion of the sample pad, or may be contained in the entire sample pad.

[0072] Specific examples of materials suitable for the sample pad include, but are not limited to, glass fiber, acrylic fiber, hydrophilic polyethylene material, dry paper, pulp, fabric, etc. Preferably, a glass fiber pad is used. The sample pad may also have the function of a conjugate pad described later. Without departing from the desired function of the present invention, the sample pad may also contain a blocking agent as needed in a manner that does not affect the reaction of the detection mechanism.

[0073] (Conjugate)

[0074] In the conjugate of the present invention, a specific binding substance or a control substance that binds to an analyte is immobilized on a label.

[0075] The label used in the present invention can be any label that can constitute a conjugate by conjugating (immobilizing) a specific binding substance such as an antibody, and can be used as a label in a method of contacting the label with a sample to detect an object (e.g., an antigen) in the sample, and examples thereof include colloidal gold particles, colloidal platinum particles, colored latex particles, and magnetic particles, among which colloidal gold and colored latex are ideal, and colloidal gold is more ideal. The particle size can be adjusted to obtain the detection sensitivity of the desired measurement object according to each type, for example, the particle size of the colloidal gold particles is preferably 20 to 100 nm, more preferably 30 to 100 nm, and most preferably 60 nm.

[0076] In the conjugate of the present invention, the region on the colloidal gold particle that is not bound to the specific binding substance may be blocked with a blocking agent.

[0077] Regarding the form of the conjugate, the conjugate may be present as a conjugate pad, that is, in a state of being contained in a dedicated pad (conjugate pad) other than the sample pad, the third pad, and the porous membrane (Type A), or as a conjugate portion in a part of the sample pad (Type B). Alternatively, the conjugate may be present as a separate conjugate reagent separated from the test piece so as to be mixed with the sample (Type C).

[0078] A test piece having a conjugate in the form of Type A will be described below.

[0079] The sample pad, conjugate pad, third pad and porous membrane are arranged in this order from upstream to downstream in the flow direction of the sample, and arranged so that the upper layer and the lower layer at least partially overlap each other. Figure 4 shown.

[0080] When a sample containing an analyte is loaded onto the sample pad of such a test piece, the analyte flows through the sample pad to the conjugate pad on the downstream side. In the conjugate pad, the analyte and the conjugate contact each other and pass through the pad, while forming a complex. Subsequently, the complex passes through a third pad disposed in contact with the lower surface of the conjugate pad and is developed in a porous membrane.

[0081] Since the porous membrane has a specific binding substance that binds to the analyte immobilized on a portion thereof, the complex is bound and immobilized on the membrane. The immobilized complex is detected by detecting absorbance, reflected light, fluorescence, magnetism, etc. derived from the labeled substance.

[0082] A test piece of the conjugate having the Form B presence will then be described.

[0083] The difference from the type A test strip is that the sample pad and the conjugate pad are integrated, that is, the sample application area and the conjugate portion are formed in a part of the sample pad.

[0084] The sample application zone is a site where a sample containing an analyte is loaded, the conjugate portion is a site containing a conjugate, and the sample application zone is located upstream of the conjugate portion.

[0085] A test piece of the conjugate having the Form C will then be described.

[0086] The difference with the A-type test strip is that there is no conjugate pad in the test strip, and the conjugate exists as a separate conjugate reagent. For example, a filter chip with a conjugate incorporated in a filter may be included. By using this filter chip to filter the sample using a filter, the conjugate retained in the filter is combined with the analyte to form a complex (aggregate). The complex can be loaded onto the same test strip as the A-type test strip in the absence of a conjugate pad to detect the analyte.

[0087] (Detection Reagents)

[0088] In the present invention, the "detection reagent" is specifically a solution containing at least a conjugate.

[0089] The detection reagent may include, for example, one or more types of stabilizers, solubilizers, etc., to maintain the conjugate in a stable state, so as to promote the specific reaction of the specific binding substance (e.g., antibody) immobilized on the conjugate with the analyte when mixed with the sample, or to quickly and effectively dissolve and fluidize the conjugate. Examples of stabilizers, solubilizers, etc. include bovine serum albumin (BSA), sucrose, casein, and amino acids.

[0090] In order to improve the detection sensitivity, the detection reagent may also contain known sensitizers and chelators, such as EDTA and EGTA, as required.

[0091] In the present specification, the term "detection" or "measurement" must be interpreted in the broadest sense, including the demonstration of the presence and / or the quantification of the analyte, and must not be interpreted in a restrictive manner in any sense.

[0092] (Sample diluent)

[0093] For the present invention, when the concentration of the analyte in the sample needs to be diluted, a diluent can be used. The diluent can be used in the following situations, such as when there is an inhibitory effect between the analyte and the specific binding substance or the reaction is accelerated, resulting in aggregation of the detection marker and impaired capillary flow, as long as the analyte concentration is maintained at a level that can react with the specific binding substance and generate a positive signal.

[0094] (Conjugate Pad)

[0095] In the present invention, "conjugate pad" refers to a pad obtained by drying a material suitable for a conjugate pad described later, in which a detection reagent that specifically reacts with an analyte is impregnated. The conjugate pad has a function of allowing the detection reagent and the analyte to form a complex when the sample passes through the conjugate pad. The conjugate pad itself can be arranged to contact a porous membrane to which a specific binding substance is fixed. Alternatively, the conjugate pad can be arranged to contact a sample pad to receive a sample passing through the sample pad via capillary flow, and then the sample is transferred to a third pad by capillary flow, which is in contact with a surface different from the contact surface of the sample pad.

[0096] Examples of suitable materials for the conjugate pad include, but are not limited to, paper, cellulose blends, nitrocellulose, polyester, acrylonitrile copolymers, glass fibers, and nonwoven fibers (eg, rayon). Preferably, a glass fiber mat is used.

[0097] The conjugate pad may contain a "control reagent" for ensuring the reliability of the immunochromatographic detection method, for example, an antibody labeled with a marker and not reactive with sample components, and a high antigen protein such as KLH (keyholelimpet hemocyanin) labeled with a marker as needed. These control reagents are components (substances) that are unlikely to be present in the sample and can be appropriately selected.

[0098] (Third pad)

[0099] In the present invention, a third pad can be added to remove reactive components and detection reagents that are not needed by the analyte in the test sample, so that the components necessary for the reaction can be successfully developed on the porous membrane fixed with the specific binding substance. For example, it is desirable to remove blood cells and insoluble blood cell parts that are components not needed for the test. The third pad can also have another function, namely, to pre-remove aggregates generated by the reaction between the analyte and the specific binding substance, which have grown to a size that prevents movement to the membrane fixed with the specific binding substance and smooth development through the membrane.

[0100] The third pad can be made of any material and in any form that allows the liquid and the components to be tested and the detection reagent to pass through. Specific examples include, but are not limited to, glass fiber, acrylic fiber, hydrophilic polyethylene material, dry paper, pulp, fabric, etc. When used to separate blood cells, the third pad can be referred to as a blood cell separation membrane. In the present invention, when whole blood is used as a sample, when the separation and removal of blood cells cannot be completed only by the sample pad, it is desirable to use a blood cell separation membrane.

[0101] (Hemagglutinating agent)

[0102] In the present invention, when whole blood is used as a sample, in addition to using the third pad, it is also desirable to use a hemagglutinating agent or a hemagglutinating agent together. Although there is no particular limitation on the hemagglutinating agent or hemagglutinating agent to be used together, known examples thereof include agglutinin, polyclonal antibodies and monoclonal antibodies, and polycationic hemagglutinating agents may also be used. Examples of known polycationic hemagglutinating agents include polybrene, polylysine, polyacrylamide and polyalanine, of which polybrene is preferred. The chemical name of polybrene is hexadimethrinebromide, which is one of the cationic polymers and has a CAS number of 28728-55-4.

[0103] The erythrocyte agglutinating agent or erythrocyte binding component can be used in the form of adding the reagent or component to a diluent for diluting a sample or directly adding it to a sample, or can be contained in a sample application area (sample pad) of an immunochromatographic test strip. In this form of use, erythrocytes in whole blood are agglutinated.

[0104] (porous membrane)

[0105] The porous membrane used in the immunochromatographic test piece of the present invention is characterized in that a specific binding substance for detection is immobilized on the surface thereof, and an opaque substrate is provided on the back surface.

[0106] Since the film is porous, the opaque substrate of the present invention disposed on the back side of the porous film is arranged to line the film and is an impermeable film. Such an opaque substrate of the present invention may also be referred to as a so-called "lining material". The substrate may be fixed on the back side of the porous film for lining, or the porous film may be laminated in advance on the upper surface of the impermeable film.

[0107] The opaque substrate of the present invention can be arranged on the back side of the porous film and can be in contact with the film through an adhesive layer, or can be in contact with the film without an adhesive layer if adhesion is ensured. When a liner sheet described later is arranged, the liner sheet is arranged below the opaque substrate (lining material) of the present invention.

[0108] In the present invention, an opaque substrate is used on the back of a porous film like a conventional transparent substrate, thereby avoiding the so-called "bleaching phenomenon" (hereinafter also referred to as bleaching), in which the corresponding position on the film appears relatively whiter than the surrounding color, as if the position has been bleached.

[0109] The thickness of the opaque substrate of the present invention (excluding adhesive layer, etc.) can be set so that the substrate can be used as a lining material for a porous membrane, and is preferably 30 μm to 200 μm, more preferably 60 μm to 140 μm, and most preferably 80 μm to 120 μm.

[0110] Opaque means not transparent or unable to see through to the other side, although complete opacity is not required, but is more preferred.

[0111] The opaque substrate is preferably a substrate having a color similar to that of the porous film, and when the porous film is white, the substrate of the present invention is preferably milky white or gray close to white, most preferably white.

[0112] The opaque substrate may be any water-impermeable opaque substrate and may be made of any material. Examples of materials include polyethylene, polyethylene terephthalate, polyester, nylon, and the like.

[0113] By placing the opaque substrate of the present invention on the back of the porous membrane, the variation of the measured value on the test line can be suppressed. Although the reason is not clear, it can be speculated that the color matching on the upper surface and the back of the membrane leads to a reduction in the uneven appearance of the surface component (nitrocellulose) or a reduction in interference caused by the reflected light of the liner placed under the lower layer of the membrane, etc., resulting in an improvement in reproducibility.

[0114] The porous membrane (hereinafter also referred to as membrane) can be made of any material. Examples include but are not limited to polyethylene, polyethylene terephthalate, nylon, glass, polysaccharides such as cellulose and cellulose derivatives, or ceramics. Specific examples may include glass fiber filter paper and nitrocellulose membrane purchased from Merck Millipore, Toyo Roshi Kaisha, GE Healthcare, etc. By appropriately selecting the pore size and structure of the porous membrane, the rate of the immune complex composed of the specific binding substance of the mark and the analyte flowing through the membrane can be controlled. Since the amount of the specific binding substance of the mark combined with the specific binding substance fixed on the membrane can be regulated by the flow velocity in the control membrane, considering the combination with other constituent materials of the immunochromatographic test piece of the present invention, it is desirable to optimize the pore size and structure of the membrane.

[0115] (Immobilization of specific binding substances on porous membranes)

[0116] Specific binding substances (e.g., antibodies to the analyte of the present invention) can be fixed on a porous membrane by well-known methods. For example, in the case of a flow-through type, the specific binding substance is adjusted to a predetermined concentration, and a constant amount of its solution is applied to the porous membrane in a certain symbol shape (e.g., a dot or +). In this case, in order to ensure the reliability of the immunochromatographic detection method, a protein or compound capable of binding to a conjugate is usually fixed at a position different from the specific binding substance bound to the analyte to form a "control line". The above-mentioned specific binding substance bound to a control reagent can be fixed at a position different from the specific binding substance bound to the analyte to form a "control line".

[0117] In the case of lateral flow (lateral-flow) type, the specific binding substance is adjusted to a predetermined concentration, and the solution is applied to the porous membrane in a linear manner using a device, etc., and the device has a mechanism that can move the nozzle in the horizontal direction while discharging the solution at a constant rate. In this case, the concentration of the specific binding substance is preferably 0.1 to 5mg / mL, more preferably 0.5 to 3mg / mL. In the case of the flow type, the amount of the specific binding substance fixed on the porous membrane can be optimized by adjusting the amount of application dripped onto the porous membrane, and in the case of the lateral flow type, the discharge rate of the nozzle of the above-mentioned device can be optimized. In particular, in the case of the lateral flow type, preferably 0.5 to 2 μL / cm.

[0118] In the present invention, the term "flow-through membrane assay" refers to a method in which a sample solution, etc. is developed to pass vertically through a porous membrane, and the term "lateral flow membrane assay" refers to a method in which a sample solution, etc. is developed to move in a direction parallel to the porous membrane.

[0119] In the present invention, regarding the application position of the specific binding substance that binds to the object on the porous membrane, in the case of lateral flow, the position can be arranged so that the detection reagent is unfolded from the conjugate pad due to capillary action and passes through the line to which the corresponding specific binding substance is applied in sequence. Preferably, the line formed by applying the specific binding substance that binds to the analyte is located upstream, and the line formed by applying the control specific binding substance is preferably located downstream thereof. In this case, it is desired that the lines are spaced apart at a sufficient distance so that the signal of the marker can be detected. Even in the case of the flow-through type, the application position of the specific binding substance that binds to the object can be arranged so that the signal of the marker can be detected.

[0120] The specific binding substance solution applied to the porous membrane can generally be prepared by using a predetermined buffer solution. Examples of the type of buffer solution include commonly used buffer solutions such as phosphate buffer solution, Tris buffer solution and Good's buffer solution. The pH of the buffer solution is preferably in the range of 6.0 to 9.5, and can be appropriately adjusted according to the properties of the specific binding substance used.

[0121] For example, a buffer solution with a pH of 8.0 can be used for the anti-cTnI antibody described later. The buffer solution may further contain salts such as NaCl, stabilizers and preservatives such as sucrose, preservatives such as ProClin, and the like. Salts include salts for adjusting ionic strength, such as NaCl, and salts added in the step of adjusting the pH of the buffer solution, such as sodium hydroxide. After the specific binding substance is fixed on the porous membrane, it can be further blocked by coating the area other than the specific binding substance fixing site with a commonly used blocking agent that is converted into a solution or vapor form. In this specification, the porous membrane to which the specific binding substance is fixed as described above is referred to as a "membrane to which the specific binding substance is fixed".

[0122] (Absorbent pad)

[0123] In the present invention, the absorption pad is a portion having liquid absorption capacity, and is used to control the spread of the sample by absorbing the sample that has moved and passed through the porous membrane. In the lateral flow type, the absorption pad can be arranged on the most downstream side of the test piece, and in the flow-through type, the absorption pad can be arranged, for example, at the lower part of the membrane to which the specific binding substance is fixed. For the absorption pad, for example, filter paper can be used; however, the present invention is not limited thereto.

[0124] (lining)

[0125] In the present invention, the liner is a solid phase for supporting the test piece, and a plastic sheet with an adhesive material attached is generally used. Examples of liner materials include, but are not limited to, polystyrene, polyester, polypropylene, vinyl chloride, etc. Although the color of the liner is not particularly limited, the color is preferably the same color as the film, and when the film is white, the liner is also preferably white.

[0126] The thickness of the lining sheet of the present invention (excluding the adhesive layer, etc.) can be adjusted so that the lining sheet can be used as a supporting member for the test piece, and is preferably from 10 μm to 600 μm, more preferably from 150 μm to 350 μm, and most preferably from 200 μm to 320 μm.

[0127] (Top film)

[0128] In the present invention, the top film refers to a sheet-like member covering the uppermost surface of the test piece, and is used to increase the mechanical strength of the porous membrane fixed with a specific binding substance and prevent the evaporation of water (drying) during the determination process. The material of the top film can be any material that can achieve the purpose, such as polyester, and the test piece can be coated with an adhesive material or can be laminated. In the present invention, it is found that by making the backing member of the film opaque, the bleaching phenomenon enhanced by covering the test piece with the top film can be weakened. Therefore, the effect of the present invention is more significant in a test piece with a top film.

[0129] The thickness of the top film of the present invention (excluding the adhesive layer etc.) can be adjusted so as to achieve the functions of protecting the film and preventing water evaporation during measurement, and is preferably 10 μm to 150 μm, more preferably 20 μm to 80 μm.

[0130] (Detection device)

[0131] Taking into account the size of the test piece, the method and position of adding the sample, the fixing position of the specific binding substance on the membrane on which the specific binding substance is fixed, the signal detection method, etc., the test piece for immunochromatography of the present invention can be stored / installed and used in a suitable container (housing), and the state of being stored / installed in this manner is called a "device". Figure 5 and Figure 6 An example of a device according to the invention is shown in a top view and in a cross-sectional view.

[0132] (other)

[0133] In this specification, "porous membrane" can be expressed as "solid phase", and specific binding substances such as antigens or antibodies are physically or chemically supported on the porous membrane, or the support state can be expressed as "fixed", "immobilized", "solid phase", "sensitized" or "adsorbed".

[0134] (sample)

[0135] In the detection method of the present invention, the "sample" containing the analyte refers to a biological sample such as blood, urine, sputum, saliva, nasal secretions, nasal swabs, throat swabs, other body fluids and feces. The biological sample can be used directly as a sample, and samples appropriately diluted or extracted and / or filtered with a diluent are also included in the sample of the present invention. Examples of blood samples include whole blood, red blood cells, plasma, serum, etc.

[0136] Blood samples also include samples collected through blood collection tubes to which anticoagulants (such as EDTA or heparin) are added when the blood is collected.

[0137] (Analyte)

[0138] The analyte of the present invention is a substance present in a biological sample, such as blood (whole blood), red blood cells, serum, plasma, urine, saliva or sputum, and examples thereof include: markers associated with inflammation, such as CRP (C-reactive protein), IgA, IgG and IgM; coagulation or fibrinolysis markers, such as fibrin degradation products (such as D-dimer), soluble fibrin, TAT (thrombin-antithrombin complex) and PIC (plasmin-plasmin inhibitor complex); markers associated with circulation, such as oxidized LD L, BNP (brain natriuretic peptide), H-FABP (cardiac fatty acid binding protein), cardiac troponin I (cTnI); metabolism-related markers such as adiponectin; tumor markers such as CEA (carcinoembryonic antigen), AFP (alpha-fetoprotein), CA19-9, CA125 and PSA (prostate-specific antigen); markers related to infectious diseases such as HBV (hepatitis B virus), HCV (hepatitis C virus), Chlamydia trachomatis and gonococci; allergen-specific IgE (immunoglobulin E), hormones and drugs. Among them, D-dimer, CRP, BNP, H-FABP, cTnI, etc., which are related to the strong desire to use whole blood as a sample, are more preferred.

[0139] (Specific binding substance)

[0140] In the present invention, examples of specific binding substances for analytes supported by insoluble carrier particles such as colloidal gold and porous membranes include proteins, peptides, amino acids, lipids, sugars, DNA, RNA, receptors and haptens, although there are no particular limitations on the size or origin (e.g., natural or synthetic) of the molecular weight, examples of which include antibodies or antigens that can be used in immunological measurement methods utilizing immune responses.

[0141] (Antibodies used in the present invention)

[0142] The antibody against the analyte used in the present invention is not limited by the preparation method, as long as the antibody reacts specifically with the analyte, and can be a polyclonal antibody or a monoclonal antibody. More preferably, the antibody is a monoclonal antibody. Generally, a hybridoma producing an antibody can be prepared by cell fusion between spleen cells of an animal immunized with the analyte as an immunogen and myeloma cells of the same species according to the method of Kohler and Milstein (see Nature, Vol. 256, p. 495 (1975)).

[0143] The antibody of the present invention can be a complete antibody molecule or a functional fragment of an antibody having antigen-antibody reactivity. The antibody can be an antibody obtained by an immunization step of a common animal (mouse, goat, sheep, etc.), and an antibody (e.g., a chimeric specific binding substance, a humanized antibody, or a fully humanized antibody) whose amino acid sequence is changed to an amino acid sequence of an animal species different from that of the animal immunized with the immunogen (analyte) by gene recombination technology. Examples of functional fragments of antibodies include fragments F(ab')2 or Fab' having antigen-antibody reactivity and single-chain antibodies (scFv). These functional fragments can be produced by treating the antibodies obtained as described above with a proteolytic enzyme (e.g., pepsin or papain).

[0144] When the antibody used in an assay method for detecting an analyte by a so-called sandwich formation method is a monoclonal antibody, in the relationship between the antibody fixed on the marker (first antibody) and the antibody fixed on the porous membrane (second antibody), when the epitope of the first antibody is monovalent, the epitope of the second antibody used is different from the epitope of the first antibody, and when the epitope of the first antibody is multivalent, the epitope of the second antibody used may be the same as or different from the epitope of the first antibody.

[0145] (Reagent test kit)

[0146] The detection kit using the immunochromatographic test piece of the present invention can be a kit including an immunochromatographic test piece, which includes at least a porous membrane and has a sample application area, a development area and a detection area with antibodies fixed thereon. The immunochromatographic test piece is characterized in that an opaque substrate is provided on the back of the porous membrane.

[0147] The detection kit may include another reagent required for detection (eg, a detection reagent containing a conjugate), a sample diluent, a test tube, a cotton swab for collecting stool, instructions, a housing for storing a test strip, and the like.

[0148] Specific embodiments of the present invention will be described below; however, these are for illustrative purposes only, and the present invention is not limited thereto.

[0149] Example

[0150] [Test Example 1] Confirmation test of the effect of avoiding bleaching phenomenon

[0151] The effect of preventing bleaching in the case of using the antibody-immobilized membrane of the present invention backed by a white substrate was compared with the case of backing by a conventional transparent substrate.

[0152] 1. Preparation of the Immunochromatographic Detection Device of the Present Invention

[0153] 1) Preparation of colloidal gold-labeled anti-cTnI monoclonal antibody (anti-cTnI antibody conjugate)

[0154] (i) Preparation of colloidal gold solution (60 nm)

[0155] To 5000 mL of purified water heated to 93° C., 10 mL of a 7% (w / v) aqueous solution of triammonium citrate was added and mixed by stirring. Subsequently, 10 mL of a 5% (w / v) aqueous solution of tetrachloroauric (III) acid was added and reacted for 10 minutes under stirring, and then the reaction solution was boiled. Subsequently, the solution was cooled in ice water to prepare a colloidal gold solution having an average particle size of 60 nm.

[0156] The colloidal gold solution having an average particle size of 60 nm was adjusted with purified water to have an absorbance of 1 OD / mL at the maximum absorption wavelength of colloidal gold.

[0157] (ii) Preparation of anti-cTnI antibody conjugate

[0158] To 200 mL of 1OD / mL 60 nm colloidal gold solution (pH 8.0), 10 mL of anti-cTnI monoclonal antibody diluted to 69.3 μg / mL with 2 mM Tris-HCl buffer solution (pH 7.0) was added and stirred at room temperature for 10 minutes. To the mixed solution of colloidal gold and antibody, 10 mL of purified water containing 0.5% (w / v) Neo Protein Saver (Toyobo, No. NPS-301) was added and stirred at room temperature for 5 minutes. Subsequently, the mixture was centrifuged at 11900 × g for 45 minutes at 10°C. After removing the supernatant, 10 mL of 0.2% (w / v) Neo Protein Saver aqueous solution was added to the obtained precipitate to suspend the conjugate, thereby obtaining an anti-cTnI antibody conjugate.

[0159] (iii) Preparation of colloidal gold solution (40 nm)

[0160] To 5000 mL of purified water heated to 73° C., 10 mL of a 5% (w / v) aqueous solution of triammonium citrate was added and mixed by stirring. Subsequently, 10 mL of a 5% (w / v) aqueous solution of tetrachloroauric (III) acid was added and reacted for 10 minutes under stirring, and then the reaction solution was boiled. Subsequently, the solution was cooled in ice water to prepare a colloidal gold solution having an average particle size of 40 nm.

[0161] The colloidal gold solution having an average particle size of 40 nm was adjusted with purified water to have an absorbance of 1 OD / mL at the maximum absorption wavelength of colloidal gold.

[0162] (iv) Preparation of colloidal gold-labeled KLH (KLH conjugate) for control line

[0163] To 200 mL of 1 OD / mL 40 nm colloidal gold solution (pH 6.1), 2.67 mL of KLH (manufactured by Sigma) dissolved in 2 mM phosphate buffer (pH 6.1) to 375 μg / mL was added, and stirred at room temperature for 10 minutes. To the mixed solution of colloidal gold and KLH, 20 mL of 10% bovine serum albumin (BSA) aqueous solution was added, and stirred at room temperature for 5 minutes. Subsequently, the mixture was centrifuged at 10 ° C for 45 minutes, and after removing the supernatant, 10.7 mL of conjugate dilution buffer (Scripps) was added to the obtained precipitate to suspend the conjugate, thereby obtaining a KLH conjugate.

[0164] 2) Preparation of conjugate pad

[0165] The conjugate solution was prepared by mixing anti-cTnI antibody conjugate (3OD), KLH conjugate (0.75OD), 0.5% Lipidure BL-1301, 0.25mg / ml Heteroblock, 2.4% lactose, 2.0% NPS, 20mM MOPS (pH 7.2), and a conjugate dilution buffer in an amount of 1 / 6 of the total amount, and a glass fiber pad (Merck Millipore) having a fixed volume was impregnated with the solution in an amount of 1.2 times its volume. The pad was dried by heating in a drying oven at 70°C for 45 minutes to obtain a conjugate pad.

[0166] 3) Preparation of anti-cTnI monoclonal antibody immobilized membrane (antibody-immobilized membrane)

[0167] For the test line, anti-cTnI monoclonal antibody was diluted to 3 mg / mL in 10 mM PB (containing 0.09% NaN3) (pH 8.0) containing 2.5% sucrose.

[0168] For the control line, rabbit anti-KLH polyclonal antibody (manufactured by Bethyl) was diluted to 1 mg / mL with 10 mM PB (containing 0.09% NaN3) (pH 7.2) containing 2.5% sucrose.

[0169] The anti-cTnI monoclonal antibody was set at 1 μL / cm at the short side of the nitrocellulose membrane lined with a transparent or white substrate shown in Table 1, and applied in a linear form using an immunochromatography dispenser "XYZ3050" (BIO DOT) to form a test line. Similarly, anti-KLH polyclonal antibody was applied at intervals of about 4 mm from the position of the test line to form a control line. The membrane was dried in an oven at 70° C. for 45 minutes to obtain a membrane immobilized with antibodies.

[0170] 4) Preparation of sample pad

[0171] The sample pad pretreatment solution was obtained by adjusting 20 mM MOPS (pH 7.2) containing 0.5% lactose and 2% polybrene.

[0172] A glass fiber mat (Lydall) was cut into a suitable size, injected with a sample pad pretreatment solution in an amount 1.5 times the volume of the mat, and dried in a drying oven at 70° C. for 45 minutes, and then used as a sample pad.

[0173] 5) Preparation of immunochromatographic test strips

[0174] The antibody-immobilized porous membrane (b-1) lined with each substrate (b-2) is fixed on the backing sheet (a), and the application portion is arranged so that the anti-cTnI antibody (c) on the upstream side is followed by the anti-KLH antibody (d), and then the blood cell separation membrane (third pad) (e) is installed on the membrane. Subsequently, the conjugate pad (f) manufactured in 2) is arranged and installed; the sample pad (g) manufactured in 4) is arranged and installed so as to overlap with the conjugate pad; and the absorption pad (h) is arranged and installed at one end on the opposite side.

[0175] Finally, the absorption pad and the surface of the membrane immobilized with the antibody were covered with the top film (i) so that a part of the membrane immobilized with the antibody was exposed. By cutting into a structure having components overlapped with each other in this way, an immunochromatographic test piece was manufactured. Figure 4 A schematic diagram of the structure of an immunochromatographic test piece is shown.

[0176] The test strip can be stored / mounted in a dedicated plastic housing in the form of an immunochromatographic test detection device. Figure 5 and Figure 6 A schematic top view and a schematic cross-sectional view of the immunochromatographic detection device (k) are shown. Reference numeral (n) indicates a sample addition window, (o) indicates a detection window, (L) and (m) indicate housings, and (j) indicates an immunochromatographic test piece.

[0177] 2. Detection or measurement by immunochromatography

[0178] To the sample pad window portion of the immunochromatographic test detection device prepared as described above, 120 μL of a plasma sample solution containing cardiac troponin I (cTnI) in a concentration range of 0 to 10 ng / mL was added, and visual observation was performed near the test line 15 minutes later to evaluate the presence or absence of bleaching. The results are shown in Table 1.

[0179] <Bleaching Evaluation Criteria>

[0180] By visually evaluating 12 or 20 samples, bleaching was considered "present" if bleaching was found even in one sample, and "absent" when no sample had bleaching.

[0181] [Table 1]

[0182]

[0183] 3. Results

[0184] In each case, when a nitrocellulose membrane lined with a transparent substrate was used as a membrane to which an antibody was immobilized, bleaching was observed shortly before the test line; however, when a nitrocellulose membrane lined with a white substrate of the present invention was used as a membrane to which an antibody was immobilized, bleaching was not observed. Thus, visibility is improved in visual evaluation of immunochromatographic tests, allowing for more accurate evaluation.

[0185] Although bleaching is determined / assessed by visual observation in this embodiment, if optical measurement is performed by using an apparatus, interference with the waveform can be avoided and the baseline can be correctly set.

[0186] [Test Example 2] Confirmation test of CV reduction effect

[0187] The variation of the measured values ​​when optical measurement was performed using the antibody-immobilized membrane of the present invention backed by a white substrate was compared with the case where a conventional transparent substrate was used as a backing.

[0188] 1. Fabrication of Immunochromatography Equipment

[0189] An immunochromatographic test piece was prepared in the same manner as in Test Example 1 except that two types of films lined with a transparent substrate (HF180, CN140) and a porous film lined with a white substrate of the present invention (CN150) were used.

[0190] 2. Detection or measurement by immunochromatography

[0191] 120 μL of plasma sample solution containing 40 or 60 pg / mL cardiac troponin I (cTnI) was added to the sample pad window of the immunochromatographic test device prepared above. After 15 minutes, the coloring amount (absorbance) of the test line was measured using the immunochromatographic reader Rapid Pia (Sekisui Medical Co., Ltd.), and the respective CV values ​​(variation) were calculated. CV refers to the coefficient of variation, which can be calculated by dividing the standard deviation by the mean. The results are shown in Figure 2. Figure 1 shown.

[0192] 3. Results

[0193] When the porous membrane lined with a white substrate of the present invention is used as a membrane on which an antibody is immobilized, the CV value is reduced compared to when a membrane lined with a transparent substrate is used as a membrane on which an antibody is immobilized, and the reproducibility of the measurement is improved.

[0194] [Test Example 3] Confirmation test of CV reduction effect (low concentration side)

[0195] A test was conducted to examine whether the variation reducing effect of Test Example 2 appears even when the analyte has a low concentration.

[0196] 1. Fabrication of Immunochromatography Equipment

[0197] An immunochromatographic test piece was prepared in the same manner as in Test Example 1 except that the transparent substrate-lined film (CN150) and two batches of the porous film (CN150) of the present invention lined with a white substrate were used.

[0198] 2. Detection or measurement by immunochromatography

[0199] The measurement was performed in the same manner as in Test Example 2 except that a plasma sample solution containing 15 pg / mL of cardiac troponin I (cTnI) was used, and the respective CV values ​​(variability) were calculated. Figure 2 shown.

[0200] 3. Results

[0201] When the porous membrane lined with a white substrate of the present invention is used as the membrane on which the antibody is immobilized, the CV value is reduced even at a low concentration of the analyte, and the reproducibility of the measurement is improved, as compared to when a membrane lined with a transparent substrate is used as the membrane on which the antibody is immobilized. Similar results were obtained even when two different batches were used.

[0202] [Test Example 4] Confirmation test of CV reduction effect (high concentration side)

[0203] A test was conducted to examine whether the variation reducing effect of Test Example 2 appears even when the analyte has a high concentration.

[0204] 1. Fabrication of Immunochromatography Equipment

[0205] An immunochromatographic test piece was prepared in the same manner as in Test Example 1 except that the transparent substrate-lined film (CN150) and the white substrate-lined porous film (CN150) of the present invention were used.

[0206] 2. Detection or measurement by immunochromatography

[0207] The measurement was performed in the same manner as in Test Example 2 except that a plasma sample solution containing 400 pg / mL of cardiac troponin I (cTnI) was used, and the respective CV values ​​(variability) were calculated. Figure 3 shown.

[0208] 3. Results

[0209] When the porous membrane lined with a white substrate of the present invention is used as a membrane on which antibodies are immobilized, the CV value is significantly reduced even at a high analyte concentration, compared with when a membrane lined with a transparent substrate is used as a membrane on which antibodies are immobilized, and the reproducibility of measurement is significantly improved.

[0210] Industrial Applicability

[0211] According to the immunochromatographic test piece of the present invention, it at least includes a porous membrane and has a sample application area, a development area and a detection area in which a specific binding substance is fixed, wherein the detection area is fixed on the surface of the porous membrane, and an opaque substrate is arranged on the back of the porous membrane, so that bleaching near the test line can be avoided.

[0212] In addition, it is possible to produce an immunochromatographic test piece that causes less variation in measured values.

[0213] According to the present invention, avoiding bleaching of the test strip not only significantly improves the visibility of visual determination, but also reduces the variation of measured values ​​in optical measurements, thereby enabling more accurate measurements in any detection method.

[0214] Reference numerals list

[0215] (a) Lining

[0216] (b) Antibody Immobilization Membrane

[0217] (b-1) Porous membrane

[0218] (b-2) Base material

[0219] (c) Anti-cTnI antibody (test line)

[0220] (d) Anti-KLH antibody (control line)

[0221] (e) Blood cell separation membrane (third pad)

[0222] (f) Conjugate pad

[0223] (g) Sample pad

[0224] (h) Absorbent pad

[0225] (i) Top membrane

[0226] (j) Immunochromatographic test strips

[0227] (k) Immunochromatographic detection device

[0228] (L) Upper shell

[0229] (m) Lower shell

[0230] (n) Sample addition window area

[0231] (o) Detection window area

Claims

1. An immunochromatographic test piece comprising at least a sample pad used as a sample application area, a blood cell separation membrane or a third pad, a porous membrane and an opaque substrate, the immunochromatographic test piece comprising a development area and a detection area, wherein the detection area is fixed on the surface of the porous membrane, and wherein the opaque substrate is provided on the back side of the porous membrane, the test piece is arranged on a lining sheet, and the lining sheet supports the test piece, the opaque substrate is a substrate having a color similar to that of the porous membrane, and the color of the lining sheet is the same as that of the porous membrane, The opaque substrate is provided as an inner lining of the porous membrane, a liner is provided below the opaque substrate, and a thickness of the opaque substrate is 80 μm or more and 120 μm or less.

2. The immunochromatographic test piece according to claim 1, wherein the opaque substrate is a white substrate.

3. The immunochromatographic test piece according to any one of claims 1 to 2, further comprising a conjugate pad of a specific binding substance labeled with colloidal gold or colored latex.

4. The immunochromatographic test piece according to claim 3, wherein the specific binding substance is an antibody. 5 . The immunochromatographic test piece according to claim 1 , wherein the immunochromatographic test piece is a test piece for detection by optical means.

6. An immunochromatographic detection kit, comprising: An immunochromatographic test piece according to any one of claims 1 to 5.

7. An immunochromatographic detection method, comprising using the following test strip: An immunochromatographic test piece comprising at least a sample pad serving as a sample application area, a blood cell separation membrane or a third pad, a porous membrane, and an opaque substrate disposed on the back of the porous membrane, wherein the porous membrane comprises at least a detection area, the test piece is arranged on a liner, and the liner supports the test piece, the opaque substrate is a substrate having a color similar to that of the porous membrane, and the color of the liner is the same as that of the porous membrane, The opaque substrate is provided as an inner lining of the porous membrane, a liner is provided below the opaque substrate, and a thickness of the opaque substrate is 80 μm or more and 120 μm or less.

8. The immunochromatographic detection method according to claim 7, wherein the opaque substrate is a white substrate.

9. The immunochromatographic detection method according to any one of claims 7 to 8, wherein the test strip further comprises a conjugate pad of a specific binding substance labeled with colloidal gold or colored latex.

10. The immunochromatographic detection method according to claim 9, wherein the specific binding substance is an antibody.

11. The immunochromatographic detection method according to any one of claims 7 to 8, comprising the step of detecting an optical parameter derived from the labeling substance by optical means.

12. The immunochromatographic detection method according to any one of claims 7 to 8, comprising the step of determining the color development derived from the labeling substance by visual observation.

13. A method for reducing the variation of measured values ​​in an immunochromatographic detection method, the method comprising using the following test strip: An immunochromatographic test piece comprising at least a sample pad used as a sample application area, a blood cell separation membrane or a third pad, a porous membrane and an opaque substrate, wherein the immunochromatographic test piece comprises a development area and a detection area, wherein the detection area is fixed on the surface of the porous membrane, and wherein The opaque substrate is provided on the back side of the porous film, the test piece is arranged on a liner, and the liner supports the test piece, the opaque substrate is a substrate having a color similar to that of the porous film, and the color of the liner is the same as that of the porous film, The opaque substrate is provided as an inner lining of the porous membrane, a liner is provided below the opaque substrate, and a thickness of the opaque substrate is 80 μm or more and 120 μm or less.

14. The method for reducing variation according to claim 13, wherein the opaque substrate is a white substrate.

15. A method for reducing bleaching of a test strip in an immunochromatographic detection method using the test strip, the method comprising using the following test strip: An immunochromatographic test piece comprising at least a sample pad used as a sample application area, a blood cell separation membrane or a third pad, a porous membrane and an opaque substrate, wherein the immunochromatographic test piece comprises a development area and a detection area, wherein the detection area is fixed on the surface of the porous membrane, and wherein the opaque substrate is provided on the back side of the porous membrane, the test piece is arranged on a lining sheet, and the lining sheet supports the test piece, the opaque substrate is a substrate having a color similar to that of the porous membrane, and the color of the lining sheet is the same as that of the porous membrane, The opaque substrate is provided as an inner lining of the porous membrane, a liner is provided below the opaque substrate, and a thickness of the opaque substrate is 80 μm or more and 120 μm or less.

16. The method for reducing bleaching of a test piece according to claim 15, wherein the opaque substrate is a white substrate.

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