Immunochromatographic test piece, immunochromatographic device, immunochromatographic kit, and method for detecting test substance

Abstract

This immunochromatographic test piece is used for detecting a test substance in a sample containing blood cells by immunochromatography and comprises: a matrix for developing a development liquid; a blood cell removal pad that is layered on the matrix and includes an addition section for the sample; and a waterproof layer layered between the matrix and the blood cell removal pad. The blood cell removal pad has a protruding section protruding further onto the matrix in the development direction of the development liquid than the waterproof layer.

Description

Immunochromatographic test piece, immunochromatographic device, immunochromatographic kit, and test substance detection method

[0001] The present invention relates to an immunochromatographic test piece, an immunochromatographic device, an immunochromatographic kit, and a method for detecting a test substance, and more particularly to an immunochromatographic test piece, an immunochromatographic device including the same, an immunochromatographic kit including at least one of these, and a method for detecting a test substance in a sample containing blood cells using at least one of these.

[0002] In order to determine whether an infectious disease is positive or negative and to identify its cause, it is necessary to detect pathogens such as bacteria and viruses from samples collected from a subject. It is also known that in order to determine whether a specific disease is positive or negative, a substance that serves as a marker for that disease can be detected from a sample such as blood. Immunoassays that utilize antigen-antibody reactions are known as methods for detecting test substances such as pathogens and markers.

[0003] Among the immunoassays, immunochromatography is known as a simple and rapid detection method. Examples of immunochromatography include a method in which a sample is developed on an immunochromatographic test strip using a porous membrane or the like as an immunochromatographic medium to which an antibody or antigen capable of binding to the analyte in the sample is immobilized. During the development process, a complex is formed between the analyte and a label capable of specifically binding to the analyte (e.g., an antibody or antigen capable of specifically binding to the analyte and labeled with a labeling substance). The complex is captured indirectly or directly by the antigen or antibody immobilized on the porous membrane or the like, and the analyte is detected by a signal such as color development by the labeling substance. This method is simple in operation, since the analyte in the sample can be detected simply by applying or dropping the sample onto the immunochromatographic test strip and allowing the sample or the complex to develop on the immunochromatographic medium by capillary action. Furthermore, since it is possible to detect test substances in a short time and visual judgment is also possible, it is widely used in research tests and clinical examinations.

[0004] However, when the sample is a sample containing blood cells, such as a whole blood specimen, problems arise such as clogging of the immunochromatography medium by the blood cells, and when red blood cells burst, the resulting coloring by hemoglobin makes it difficult to easily visually confirm the signal from the labeling substance. Therefore, when the sample contains blood cells, it is necessary to remove the blood cells in advance, and in the case of the immunochromatographic test strip, for example, a method is used in which a member having a blood cell removal function is provided on the test strip itself.

[0005] For example, Japanese Patent Laid-Open Publication No. 2002-350428 (Patent Document 1) describes that, for the purpose of suppressing hemolysis of red blood cells, a fiber-containing layer that retains red blood cells and separates plasma or serum from whole blood is made of glass fibers coated with propanol and / or acrylamide having butoxy groups.

[0006] Furthermore, as an example of a test strip structure that aims to eliminate the influence of blood cells, Japanese Patent Laid-Open Publication No. 2013-181870 (Patent Document 2) describes an immunochromatographic test strip that includes a chromatography medium having a determination unit supported on an impermeable substrate, and a specimen processing unit upstream of the chromatography medium to which a sample is added, and the specimen processing unit has a structure in which a sample pad to which the sample is added, a blood cell separation membrane to separate blood cells, and a conjugate pad containing a labeled substance are stacked in this order with some offset.

[0007] Furthermore, for example, JP 2011-522228 A (Patent Document 3) describes a test strip used in a method for determining blood type by dropping a blood sample onto a test strip coated and immobilized with anti-N blood group antibodies, dropping a cleaning solution onto one end of the test strip, and then determining the blood type based on whether agglutinated red blood cells remain in the reaction spot. The test strip has a cleaning pad, a sample application pad, a buffer pad, and a water-absorbing pad attached in this order to a lining, and waterproof tape attached to the upper surface of the overlapping edge of the cleaning pad and the sample application pad to prevent the cleaning solution from spreading from the surface of the sample application pad.

[0008] Furthermore, for example, JP 2017-509889 A (Patent Document 4) describes a liquid-guiding element for a lateral flow test device, and in the case of a blood sample, describes a small amount of sample, and describes an improved configuration to reduce the required volume of sample, in which each member constituting the liquid-guiding element is provided with a backing layer, and the backing layer is oriented so as to be placed on the upper or lower surface of the liquid-guiding element as the center.

[0009] Japanese Patent Application Laid-Open No. 2002-350428 Japanese Patent Application Laid-Open No. 2013-181870 Special Publication No. 2011-522228 Special Publication No. 2017-509889

[0010] Conventionally, to remove blood cells from a sample containing blood cells on an immunochromatographic test strip, it has been necessary to use a specific substrate for the component with blood cell removal function, or to position the sample application section or the component with blood cell removal function upstream of the strip-shaped test strip so that blood cells are removed during the development process, as described above. Furthermore, for example, when an enzyme is used as a labeling substance, a signal is detected by the reaction between the enzyme and a substrate. For highly accurate detection, the reaction between the enzyme and the substrate preferably occurs after a complex of the label containing the enzyme and the analyte is captured by a capture antibody or antigen. However, in conventional structures in which the sample application section is upstream of the test strip, if the development of the complex and the substrate is performed in a single operation, the capture of the complex and the enzyme-substrate reaction cannot be performed with a time lag, resulting in a problem of reduced accuracy when applied to enzyme immunoassays.

[0011] The present invention has been made in consideration of the above-mentioned problems, and aims to provide an immunochromatographic test piece that can easily detect a test substance in a sample containing blood cells, such as a whole blood specimen, and that can also perform enzyme immunoassays with high accuracy, an immunochromatographic device equipped with the same, an immunochromatographic kit that includes at least one of these, and a method for detecting a test substance in a sample containing blood cells using at least one of these.

[0012] To solve the above-mentioned problems, the present inventors conducted extensive research into test strips used for immunochromatographic detection of analytes in samples containing hemocytes. As a result, they discovered a structure in which a waterproof layer is sandwiched between a matrix, an immunochromatographic medium for developing a developing solution, and a hemocyte removal pad for removing hemocytes, and the hemocyte removal pad is slightly protruding beyond the waterproof layer in the direction of development of the developing solution, with the matrix and the hemocyte removal pad contacting only at this protruding portion. By adding a sample to the hemocyte removal pad, surprisingly, despite this extremely simple structure, hemocytes are removed from the sample before contact with the developing solution that develops the matrix, sufficiently suppressing migration of hemocytes into the matrix and enabling easy detection of the analyte. Furthermore, this structure allows the sample addition section and the developer supply section for supplying the developer solution to be installed in separate locations. Therefore, for example, by placing the application section holding the label in the center of the test piece, and by placing a developer supply section that supplies developer and a substrate zone containing a substrate to be eluted in the developer upstream of the test piece, the complex of the test substance and the label can first be captured by a capture antibody or antigen (capturer) downstream of the application section, and then the substrate that arrives later can be reacted with the enzyme contained in the label, and therefore it has been found that highly accurate enzyme immunoassays can be easily performed, leading to the completion of the present invention.

[0013] That is, the present invention relates to an immunochromatographic test strip, an immunochromatographic device including the same, an immunochromatographic kit including these, and an analyte detection method using these, and more specifically provides the following: [1] A test strip used for immunochromatographic detection of an analyte in a sample containing blood cells, comprising: a matrix for developing a developing solution; a blood cell removal pad including a sample application area layered on the matrix; and a waterproof layer layered between the matrix and the blood cell removal pad, wherein the blood cell removal pad has a protruding portion that protrudes onto the matrix beyond the waterproof layer in the direction of development of the developing solution. [2] The immunochromatographic test strip according to [1], wherein the protruding portion of the blood cell removal pad has a length of 1 to 4 mm. [3] The immunochromatographic test strip according to [1] or [2], wherein the blood cell removal pad is a glass fiber membrane. [4] The immunochromatographic test strip according to any one of [1] to [3], which comprises a capturer-immobilizing section on the matrix downstream of the blood cell removal pad, to which a capturer capable of capturing a test substance is immobilized. [5] The immunochromatographic test strip according to any one of [1] to [4], which comprises a label-containing section that elutably holds a label in which a labeling substance is bound to an antibody or antigen that can bind to the test substance. [6] The immunochromatographic test strip according to [5], in which the labeling substance is an enzyme. [7] The immunochromatographic test strip according to [6], in which the developing solution contains a substrate for the enzyme, or in which a substrate zone is provided on the matrix upstream of the capturer-immobilizing section that elutably holds the enzyme substrate, or in which a section for adding the enzyme substrate is provided on the matrix. [8] The immunochromatographic test piece according to any one of [5] to [7], wherein a label-containing pad including the label-containing portion is laminated on the blood cell removal pad, or the blood cell removal pad includes the label-containing portion. [9] An immunochromatographic device used for immunochromatographic detection of a test substance in a sample containing blood cells, the immunochromatographic device comprising the immunochromatographic test piece according to any one of [1] to [8].

[10] A kit used for immunochromatographic detection of an analyte in a sample containing blood cells, the kit comprising the immunochromatographic test strip described in any one of [1] to [8] or the immunochromatographic device described in [9].

[11] A method for immunochromatographic detection of an analyte in a sample containing blood cells, the method comprising: a matrix for developing a developing solution; a blood cell removal pad including a sample application section laminated on the matrix; and a waterproof layer laminated between the matrix and the blood cell removal pad, wherein the blood cell removal pad has an overflowing section that overflows onto the matrix beyond the waterproof layer in the direction of development of the developing solution, the method comprising the steps of: adding the sample to the application section; and spreading the developing solution on the matrix, the method comprising: bringing the sample into contact with the developing solution at the overflowing section of the blood cell removal pad.

[0014] While the reason why the above-mentioned object is achieved by the configuration of the present invention is not entirely clear, the inventors speculate as follows. Specifically, in the immunochromatographic test strip of the present invention, when a sample containing blood cells is applied from above to the application section of the blood cell removal pad, the presence of a waterproof layer on the underside of the blood cell removal pad prevents the sample from migrating vertically into the matrix below the blood cell removal pad. Furthermore, the blood cell removal pad and the waterproof layer are configured such that the blood cell removal pad has a protruding portion that protrudes beyond the waterproof layer onto the matrix in the direction of development of the developing solution. Therefore, the applied sample moves in the direction of development of the developing solution moving within the matrix, and in a direction parallel to this direction. Therefore, the sample is filtered to remove blood cells while moving within the blood cell removal pad, and only the sample from which blood cells have been removed migrates from the protruding portion at the tip of the blood cell removal pad into the matrix. The inventors speculate that this allows for simple and efficient suppression of blood cell migration into the matrix, enabling easy and highly accurate detection of the analyte.

[0015] According to the present invention, it is possible to provide an immunochromatographic test piece that can easily detect a test substance in a sample containing blood cells, such as a whole blood specimen, and that can also perform enzyme immunoassays with high accuracy, an immunochromatographic device equipped with the same, an immunochromatographic kit that includes at least one of these, and a method for detecting a test substance in a sample containing blood cells using at least one of these.

[0016] 1 is a schematic cross-sectional view showing one embodiment of the immunochromatographic test strip of the present invention (a: immunochromatographic test strip 101, b: immunochromatographic test strip 102). FIG. 2 is a schematic plan view showing one embodiment of the immunochromatographic test strip of the present invention (immunochromatographic test strip 103). FIG. 3 is a schematic cross-sectional view of FIG. 2 showing one embodiment of the immunochromatographic test strip of the present invention (immunochromatographic test strip 103). FIG. 4 is a schematic cross-sectional view of FIG. 2 showing one embodiment of the immunochromatographic test strip of the present invention (immunochromatographic test strip 201). FIG. 5 is a schematic plan view of one embodiment of the immunochromatographic device of the present invention (immunochromatographic device 202). FIG. 6 is a schematic cross-sectional view of FIG. 5 showing one embodiment of the immunochromatographic device of the present invention (immunochromatographic device 202). 1 shows the appearance of the determination window (detection zone 8 and development confirmation zone 11) upon visual confirmation when a detection test for a test substance (antigen A and antibody B) was conducted using each of the immunochromatographic devices prepared in Examples 1 and 3. 2 shows the appearance of the determination window (detection zone 8 and development confirmation zone 11) upon visual confirmation and the evaluation results when a comparison test was conducted for each of the immunochromatographic devices prepared in Examples 1 to 3 (top row) and Comparative Examples 1 to 3 (bottom row) using a whole blood sample from a healthy person (negative sample) at each drop amount. 3 shows a conceptual diagram showing positions a to c of each label-containing portion in the immunochromatographic device prepared in "(4-1) Preparation of an immunochromatographic device" in "(4) Investigation of an immunochromatographic device." This figure shows the appearance and evaluation results of the judgment window (detection zone 8 and development confirmation zone 11) when visually inspected when a detection test for a test substance (antigen C) was conducted using each immunochromatography device prepared in Examples 1-1, 1-2, 3-1, and 3-2.

[0017] The present invention will be described in detail below by way of examples of preferred embodiments, with reference to the drawings where appropriate, but the present invention is not limited thereto. In the following description and drawings, the same or corresponding elements are designated by the same reference numerals, and redundant description will be omitted.

[0018] <Immunochromatographic test strip, immunochromatographic device> The present invention provides an immunochromatographic test strip used for immunochromatographic detection of an analyte in a sample containing blood cells, comprising: a matrix for developing a developing solution; a blood cell removal pad including a sample application area laminated on the matrix; and a waterproof layer laminated between the matrix and the blood cell removal pad, wherein the blood cell removal pad has a protruding portion that protrudes beyond the waterproof layer onto the matrix in the direction of development of the developing solution. The present invention also provides an immunochromatographic device comprising the immunochromatographic test strip of the present invention.

[0019] 1 to 3 are schematic cross-sectional or plan views showing one embodiment of an immunochromatographic test strip of the present invention, and FIGS. 4 to 6 are schematic cross-sectional or plan views showing one embodiment of an immunochromatographic device of the present invention. In the following description, the left side of FIGS. 1 to 6 (the upstream side in the developing direction 4 of the developer) will be referred to as the "upstream" side of the immunochromatographic test strip, and the opposite side will be referred to as the "downstream" side. Note that FIGS. 1 to 6 illustrate a lateral flow immunochromatographic strip as an example of one embodiment of the immunochromatographic test strip, but the immunochromatographic test strip of the present invention is not limited to the configuration shown in FIGS. 1 to 6.

[0020] (Sample) In the present invention, the sample to be detected is a sample containing blood cells. However, this does not exclude the use of samples that do not contain blood cells. Examples of blood cells include red blood cells, white blood cells, and platelets. Preferably, the sample contains at least red blood cells. Such a sample is not particularly limited as long as it contains blood cells, and may be, for example, a blood cell suspension, but is typically a whole blood sample. Examples of the whole blood sample include whole blood samples collected from humans and non-human mammals (preferably humans). Furthermore, the sample may be, as necessary, a whole blood sample diluted or suspended in a diluent. Examples of the diluent include buffers (phosphate buffer, Tris buffer, Good's buffer, borate buffer, etc.), and may contain one or more surfactants, blood coagulation inhibitors, etc., as long as they do not inhibit the respective antigen-antibody reactions.

[0021] (Test Substance) In the present invention, the "test substance" to be detected is not particularly limited, and examples thereof include substances that can be contained in the whole blood sample, such as pathogens such as bacteria, protozoa, fungi, and viruses, and substances derived therefrom, such as nucleic acids, sugar chains, and proteins, as well as antibodies against these substances; and substances that serve as markers for specific diseases (proteins, polysaccharides, nucleic acids, etc.).

[0022] (Detection) In the present invention, the analyte is detected by immunochromatography, which utilizes an antigen-antibody reaction between the analyte and an antibody or antigen (sometimes referred to as a "detector" in this specification) capable of binding to the analyte. When the analyte is an antibody, the detector can be the antigen (including a substance that exhibits the same properties as the antigen in immunoassay (e.g., a partial peptide containing an epitope of the antigen, a similar structure (analog), a derivative, a modified product, etc.)) or an antibody capable of binding to the antibody. When the analyte is a substance other than an antibody, the detector can be an antibody capable of binding to the analyte.

[0023] In the present invention, the term "antibody" includes not only a complete antibody but also an antibody fragment (e.g., Fab, Fab', F(ab')). ２, Fv, single-chain antibodies, diabodies, etc.), and minibodies to which the variable regions of antibodies are bound. When the detector is an antibody, the antibody may be a polyclonal antibody or a monoclonal antibody. Such antibodies can be produced by appropriately adopting and improving a conventionally known production method or a method based thereon, depending on the target analyte, and commonly available antibodies may also be used as appropriate.

[0024] In the present invention, "detection of an analyte" includes detection to confirm the presence or absence of the analyte and quantification or semi-quantification of the amount of the analyte. In immunochromatographic test strips, detection of an analyte is typically performed by using a label in which a labeling substance is bound to the detector, allowing the label to bind to the analyte via the detector, detecting a signal derived from the labeling substance contained in the formed complex, and quantifying its intensity as needed, which is preferred in the present invention. If necessary, the success or failure of detection can be determined, for example, by detecting a signal in the development confirmation zone described below. In the present invention, "signal" includes color development (color development), decolorization, quenching, reflected light, luminescence, fluorescence, radiation from a radioisotope, etc., and includes signals that can be confirmed with the naked eye as well as signals that can be confirmed using a detection method or device appropriate for the type of signal.

[0025] The labeling substance can be any substance used as a labeling substance in known immunoassay methods or methods similar thereto, without any particular limitation. Examples include enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase; radioisotopes such as iodine, tritium, and carbon; luminescent substances such as acridinium derivatives; fluorescent substances such as europium; fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); low-molecular-weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); gold particles; latex; dinitrophenyl phosphate (DNP); and digoxigenin (DIG). These may be used alone or in combination of two or more. Among these, enzymes are preferred as the labeling substance, from the viewpoint of suitable application of the immunochromatographic test strip of the present invention. When an enzyme is used as the labeling substance, various signals generated by the reaction between the enzyme and the substrate can be detected by using a chromogenic substrate, fluorescent substrate, chemiluminescent substrate, or the like as a substrate for the enzyme.

[0026] The label may be one in which the detector and labeling substance are directly or indirectly bound, and may further include a water-soluble carrier that supports the detector and labeling substance, etc. Examples of the water-soluble carrier include sugars such as dextran, aminodextran, Ficoll (trade name), dextrin, agarose, pullulan, various celluloses (e.g., hemicellulose and lignin), chitin, chitosan, and modified forms thereof (e.g., hydrazinated dextran), proteins such as β-galactosidase and thyroglobulin, polypeptides, DNA, hemocyanin, and amino acids such as polylysine, and the water-soluble carrier may be one type or a combination of two or more types thereof.

[0027] The binding of the detector and the labeling substance can be appropriately performed by a conventionally known method or a method based thereon. The detector and the labeling substance may be directly bound via an active group or the like, or indirectly bound via an oligopeptide, a linker, or the like. Alternatively, a biotin-avidin system (for example, a detector is biotinylated, and an avidinated labeling substance is reacted therewith, and the labeling substance is bound to the detector by utilizing the interaction between biotin and avidin) may be used. The amounts of the detector, labeling substance, and, if necessary, the water-soluble carrier in the labeling substance can be appropriately adjusted. Furthermore, commonly available labels may be used as such labels.

[0028] Furthermore, in detecting a test substance using an immunochromatographic test strip, a secondary label may be bound to the detector, rather than a label, and then the detector may be detected. Here, the term "secondary label" refers to a label that can bind to the detector and that includes the label. Examples of such secondary labels include an antibody (e.g., a secondary antibody) that can bind to the detector bound to the label, and protein G or protein A bound to the label.

[0029] (Matrix) The immunochromatographic test strip of the present invention comprises a matrix (matrix 1 in Figures 1 to 4) that develops the developing solution. The matrix according to the present invention is an insoluble carrier that functions as an immunochromatographic medium and a stationary phase. The matrix supports the blood cell removal pad and waterproof layer described below as the main body of the immunochromatographic test strip, and, if necessary, also serves as an antibody-containing membrane including the capture body immobilizing portion, label-containing portion, development confirmation zone, etc. described below, and / or a substrate-containing membrane including the substrate zone, etc. described below.

[0030] The matrix of the present invention is not particularly limited, and any matrix conventionally used as an immunochromatography medium can be used as appropriate, but a porous membrane is preferred, such as a nitrocellulose membrane, a nitrocellulose mixed ester membrane, a cellulose membrane, an acetylcellulose membrane, a polysulfone membrane, a polyethersulfone membrane, a nylon membrane, glass fiber, a nonwoven fabric, a cloth, or a laminate of two or more of these, of which a nitrocellulose membrane is preferred. The migration speed of the developing solution developed in such a matrix can be changed by the material, size, pore size, and distribution of the porous membrane, and can be appropriately adjusted depending on the amount of sample, the concentration of the analyte, the purpose of detection, etc.

[0031] The shape of the matrix, i.e., the shape of the immunochromatographic test strip of the present invention, is not particularly limited and may be, for example, a circular shape in which the developer is spread concentrically, or a strip (long, thin piece) in which the developer is spread with one end upstream and the other end downstream, with the strip being preferred. The size of the matrix is ​​not particularly limited and may be, for example, a strip (long, thin piece) with a width of approximately 3 to 10 mm and a length of approximately 30 to 100 mm. The thickness of the matrix is ​​also not particularly limited and may be, for example, in the range of 100 μm to 1 mm. In this specification, the distance in the direction in which the developer is spread is referred to as "length," and when the immunochromatographic test strip is strip-shaped, the size of the short side of the matrix or a side parallel to it is referred to as "width," and the size of the long side of the matrix or a side parallel to it is referred to as "length."

[0032] (Blood Cell Removal Pad) The immunochromatographic test piece of the present invention includes a blood cell removal pad (blood cell removal pad 2 in FIGS. 1 to 4) on the matrix. The blood cell removal pad according to the present invention has the function of filtering and removing blood cells from the sample.

[0033] The blood cell removal pad according to the present invention is not particularly limited as long as it can filter blood cells from the sample, but is preferably a porous membrane, and examples thereof include a nitrocellulose membrane, a nitrocellulose mixed ester membrane, a cellulose membrane, an acetylcellulose membrane, a polysulfone membrane, a polyethersulfone membrane, a nylon membrane, a glass fiber membrane, a nonwoven fabric, and a cloth, and among these, a glass fiber membrane is preferred.

[0034] The conditions under which the blood cell removal pad can filter and remove blood cells from the sample are, for example, preferably a particle retention capacity of 2 to 3.5 μm, more preferably 2.2 to 3 μm, and even more preferably 2.2 to 2.8 μm. In the present invention, the "particle retention capacity" refers to the average particle size (μm) of particles (blood cells) that are retained within the porous membrane at a rate of 98% or more, and can be measured, for example, by flow cytometry.

[0035] Furthermore, the blood cell removal pad according to the present invention preferably has a flow rate of 20 to 50 s / 4 cm, more preferably 20 to 46 s / 4 cm, and even more preferably 25 to 46 s / 4 cm. In the present invention, the "flow rate" of a porous membrane refers to the speed at which water passes through the porous membrane from the bottom to the top (the time it takes to pass through a 4 cm thick porous membrane: s / 4 cm) under atmospheric pressure at a temperature of 20°C and a humidity of 50%, and is preferably the average value of multiple measurements.

[0036] Furthermore, the blood cell removal pad according to the present invention has a water absorption capacity of 30 to 120 mg / cm ２ It is also preferable that the density is 40 to 120 mg / cm ２ In the present invention, the "water absorption" of the porous membrane is defined as the amount of water absorbed by the porous membrane when the temperature is 20°C, the humidity is 50%, and the amount of water absorbed by the porous membrane is 1 cm ２ The absorbed water content indicates the amount of water absorbed into the porous membrane, and can be obtained by measuring the mass of the porous membrane before and after water absorption and calculating the difference therebetween (preferably the average value of measurements taken multiple times).

[0037] The size of the blood cell removal pad according to the present invention can be adjusted appropriately depending on the amount of sample, the concentration of the test substance, the purpose of detection, etc., but when the matrix is ​​in the form of a strip, it preferably has a width of 3 to 10 mm and a length of 10 to 30 mm, and more preferably has a width of 3 to 10 mm and a length of 10 to 20 mm.

[0038] The thickness of the hemocyte removal pad is preferably 240 to 1000 μm, more preferably 450 to 950 μm, and even more preferably 480 to 930 μm. In the present invention, the "thickness" of the porous membrane is preferably measured with a vernier caliper or the like under atmospheric pressure at a temperature of 20°C and a humidity of 50%, and is preferably the average value of multiple measurements. The thickness of the hemocyte removal pad can be appropriately adjusted by the amount of sample added, etc. For example, when the amount of sample added per unit area is 20 to 60 μL / cm, ２ In this case, the amount of sample added is preferably 240 to 600 μm, and the amount of sample added is preferably 60 to 90 μL / cm ２ In this case, the amount of sample added is preferably 600 to 900 μm, and the amount of sample added is 90 to 110 μL / cm ２ In this case, it is preferably 900 to 1000 μm.

[0039] (Application Portion) The blood cell removal pad according to the present invention includes a sample application portion (application portion 5 in FIGS. 1 to 6) as a site for applying or dropping the sample onto the immunochromatographic test piece.

[0040] In the blood cell removal pad, the application section is located on the upper surface of the blood cell removal pad. The application section is preferably located on the upstream side within the blood cell removal pad, more preferably within 15 mm from the upstream end of the blood cell removal pad, and is further preferably located within 50% of the total area of ​​the blood cell removal pad on the upstream end side, and even more preferably within 32% of the total area of ​​the blood cell removal pad.

[0041] There is no particular limitation on the size of the added portion according to the present invention, but when the matrix is ​​in the form of a strip, the size may be, for example, in the range of 1 to 10 mm in width and 3 to 15 mm in length.

[0042] (Waterproof Layer) The immunochromatographic test piece of the present invention includes a waterproof layer (waterproof layer 3 in Figures 1 to 4) between the matrix and the hemocyte removal pad. The waterproof layer according to the present invention has the function of interrupting the migration of the sample and developer between the matrix and the hemocyte removal pad, and the function of conducting the sample present on the waterproof layer downstream of the hemocyte removal pad.

[0043] The waterproof layer according to the present invention is not particularly limited as long as it is a substrate that is impermeable to the sample and the developing solution, but examples thereof include films made of polyester, polyethylene, polyethylene terephthalate, polypropylene, and laminates of two or more of these, and among these, polyester films are preferred.

[0044] The size of the waterproof layer according to the present invention need only be the same as or larger than the hemocyte removal pad, so that the migration of the sample and developing solution between the matrix and the hemocyte removal pad can be interrupted except for the protruding portion described below, i.e., so that the hemocyte removal pad and the matrix do not come into direct contact except for the protruding portion. This size can be adjusted appropriately depending on the size of the hemocyte removal pad, but when the matrix is ​​in a strip shape, it is preferably 3 to 10 mm wide and 8 to 28 mm long, and more preferably 3 to 10 mm wide and 8 to 18 mm long. The thickness of the waterproof layer is preferably 0.0005 to 1 mm, and more preferably 0.0005 to 0.1 mm.

[0045] (Protruding portion) In the immunochromatographic test piece of the present invention, the blood cell removal pad and the waterproof layer are laminated such that the blood cell removal pad protrudes onto the matrix in the developing direction of the developing solution more than the waterproof layer; in other words, the blood cell removal pad has a protruding portion (protruding portion 6 in Figure 1) that protrudes onto the matrix in the developing direction of the developing solution more than the waterproof layer.

[0046] FIG. 1 shows schematic cross-sectional views of immunochromatographic test strips 101 and 102 as embodiments of the immunochromatographic test strip of the present invention. In the immunochromatographic test strip of the present invention, the blood cell removal pad 2 and the waterproof layer 3 may be the same size or the waterproof layer 3 may be larger (i.e., the blood cell removal pad 2 and the matrix 1 may be separated by the waterproof layer 3 except for the protruding portion 6). For example, as shown in FIG. 1( a), their upstream ends (upstream of the developing solution's developing direction 4) may overlap, or as shown in FIG. 1( b), their upstream ends (upstream of the developing solution's developing direction 4) may be offset. As shown in FIG. 1, it is preferable that at least a portion of the protruding portion 6 directly contacts the matrix 1; however, complete contact is not necessary as long as the sample can be moved by the surface tension of the liquid, etc.

[0047] The length of the protruding portion is preferably 1 to 4 mm, more preferably 2 to 4 mm. The length of the protruding portion is also preferably such that the area of ​​the protruding portion on the downstream end of the blood cell removal pad accounts for 13 to 34% of the total area of ​​the blood cell removal pad, more preferably 13 to 20%. The length of the protruding portion does not take into account the curvature of the blood cell removal pad, i.e., when the immunochromatographic test strip is placed upside down on a flat surface with the blood cell removal pad and waterproof layer in that order from the bottom, the downstream end of the waterproof layer is set to 0 mm, and the length from there to the downstream end of the blood cell removal pad protruding from that point is indicated. Furthermore, when the matrix is ​​strip-shaped, the width of the protruding portion is preferably 3 to 10 mm. In Figures 1 to 4, the shape of the protruding portion 6 is preferably rectangular, conforming to the shape of the matrix. However, the shape of the protruding portion according to the present invention is not limited to this and may be any shape, such as semicircular or semipolygonal. In this case, the width of the protruding portion is defined as the maximum vertical length from the downstream end of the waterproof layer, which is set to 0 mm, to the downstream end of the blood cell removal pad.

[0048] (Capturer-immobilizing section) The immunochromatographic test strip of the present invention preferably includes a capturer-immobilizing section (capturer-immobilizing section 8 in Figures 2 to 6) on the matrix downstream of the blood cell removal pad, in which a capturer capable of capturing an analyte is immobilized on the matrix. The capturer-immobilizing section functions as a detection zone of the immunochromatographic test strip of the present invention. This allows, for example, the analyte to be captured by the capturer in the capturer-immobilizing section, and then reacted with the label that recognizes the analyte or the capturer-analyte complex, thereby enabling detection of the analyte captured by the capturer based on a signal corresponding to the type of label.

[0049] In another embodiment, more preferably, a complex between the analyte and the label is first formed, and then the complex is captured by a capturer at the capturer-immobilizing portion to form a capturer-analyte-labeled complex. The complex captured by the capturer, i.e., the analyte, is detected based on a signal corresponding to the type of label. In this case, when the complex between the analyte and the label migrates to the capturer-immobilizing portion, the complex is captured by the capturer immobilized at the capturer-immobilizing portion, and the labeled substance constituting the complex accumulates at the capturer-immobilizing portion. As a result, the capturer-immobilizing portion exhibits a signal due to the accumulation of the labeled substance, and by detecting this signal, the analyte in the sample can be detected.

[0050] In the present invention, a "capture body capable of capturing a analyte" refers to a substance capable of binding to the analyte or a complex of the analyte and a label, and capable of capturing the analyte or the complex. Such capture bodies are typically antibodies or antigens capable of binding to the analyte; or antibodies capable of specifically binding to the binding site between the analyte and the antibody or antigen contained in the label. The antibody or antigen contained in such a capture body may be the same as or different from the antibody or antigen listed as the detector in the label, but it is preferable that they do not compete with each other in binding to the analyte, depending on the recognition site of the analyte.

[0051] 2 to 6, capture agent-fixing portion 8 is formed in a line shape, but the shape of the capture agent-fixing portion according to the present invention is not limited to this and can be any shape such as a circle, a polygon, etc. Among these shapes, the shape of the capture agent-fixing portion according to the present invention is preferably a line shape, and when the matrix is ​​in a strip shape, a line shape having a width of 3 to 10 mm and a length of 0.5 to 3 mm is more preferable.

[0052] Furthermore, the capture-body-immobilizing portion according to the present invention may be plural, depending on the type of analyte and the detection method. For example, when label A containing antibody a capable of binding to analyte a and label B containing antibody b capable of binding to analyte b are used as labels, the capture-body-immobilizing portion A to which an antibody capable of binding to analyte a is immobilized and the capture-body-immobilizing portion B to which an antibody capable of binding to analyte b is immobilized may be provided correspondingly.

[0053] The method for immobilizing the capturer on the capturer-immobilizing portion of the present invention is not particularly limited, and any conventionally known method or a method equivalent thereto can be appropriately adopted, such as a physical adsorption method or a method of binding to the matrix by covalent bonding, and blocking may be performed as necessary. The amount of the capturer immobilized on the capturer-immobilizing portion of the present invention can be appropriately adjusted depending on the amount of sample, the type of detector contained in the label, the sensitivity of the capturer to the analyte, the purpose of detection, etc.

[0054] (Label-Containing Portion) In the present invention, the sample may be mixed with the label in advance and then added to the application portion, but it is preferable that the immunochromatographic test piece of the present invention comprises a label-containing portion that holds the label in an elutable manner, and it is more preferable that the label-containing portion be provided upstream of the capture body-immobilizing portion. In these cases, when the sample comes into contact with the label-containing portion, the label is eluted, and if the analyte is present in the sample, an antigen-antibody reaction between the analyte and a detector constituting the label forms a complex (immune complex) in which the analyte is bound to the label.

[0055] The label-containing portion may be, as in Examples 1 to 3 below, a label-containing portion contained in the hemocyte removal pad, with a portion of the hemocyte removal pad also serving as the label-containing portion; a label-containing pad in which the label is elutable and held in a separate water-absorbent material, which is laminated on the upper or lower surface of the hemocyte removal pad; or a label-containing pad provided at a separate location downstream from the hemocyte removal pad. The water-absorbent material is preferably a porous material, such as the porous membranes mentioned above for the hemocyte removal pad. Furthermore, when provided at a separate location from the hemocyte removal pad, the label may be elutable and held in the matrix, forming the label-containing portion. Furthermore, when the label-containing pad is laminated on the hemocyte removal pad, the label-containing portion is preferably located in a position overlapping with the application portion. When the label-containing pad is laminated on the upper surface of the hemocyte removal pad, the sample may be added from the upper surface of the label-containing pad. Among these, when the immunochromatographic test piece of the present invention comprises a label-containing portion, it is preferable that a label-containing pad is laminated on the upper surface of the blood cell removal pad or that the blood cell removal pad contains a label-containing portion, and it is more preferable that the blood cell removal pad contains a label-containing portion.

[0056] The size of the label-containing portion is not particularly limited, but when the matrix is ​​strip-shaped, it may be, for example, 1 to 10 mm wide and 3 to 30 mm long. When the label-containing portion is a label-containing pad separate from the hemocyte removal pad, its thickness may be, for example, 0.3 to 2 mm. Furthermore, when the hemocyte removal pad includes a label-containing portion, the label-containing portion is preferably located in a position overlapping with the application portion (more preferably, the application portion doubles as the label-containing portion). In this case, the label-containing portion is preferably located upstream within the hemocyte removal pad, more preferably within 15 mm from the upstream end of the hemocyte removal pad, and even more preferably within 50% and even more preferably within 32% of the total area of ​​the hemocyte removal pad on the upstream side. Furthermore, the label-containing portion is preferably applied or impregnated from the upper surface of the hemocyte removal pad. The shape of the label-containing portion is not particularly limited, and may be, for example, dotted or linear.

[0057] The method for retaining the label in the label-containing portion is not particularly limited as long as it allows the label to be eluted into the sample when it is developed, and examples thereof include a method in which a solution containing the label is applied to or impregnated into the above-mentioned hemocyte removal pad or label-containing pad or matrix, followed by drying. Examples of the solution containing the label include buffer solutions, and examples of the buffer solution include buffer solutions that can be adjusted to a pH suitable for the target antigen-antibody reaction, more specifically, phosphate buffer, Tris buffer, Good's buffer, borate buffer, etc. The amount of the label retained in the label-containing portion can be adjusted as appropriate depending on the amount of sample, the sensitivity of the detector contained in the label to the analyte, the purpose of detection, etc.

[0058] (Developer Supply Unit) The immunochromatographic test strip of the present invention preferably includes a developer supply unit (developer supply unit 7 in FIGS. 2 to 6) for supplying a developer for performing immunochromatography. The developer supply unit may be supplied by dropping the developer or from a device containing the developer, such as a developer tank described below. The developer supply unit according to the present invention is preferably located at the most upstream position of the matrix.

[0059] The developer supply part may be integrated with the matrix, but is preferably a separate member made of a water-absorbent material, such as the developer pad 7 (FIGS. 4 to 6). The base material for such a developer supply part is not particularly limited, and examples thereof include water-absorbent materials such as cellulose filter paper, nonwoven fabric, cloth, and cellulose acetate membrane.

[0060] The size of the developer supply part is not particularly limited, but when the matrix is ​​in the form of a strip, the size may be, for example, in the range of 1 to 10 mm in width and 10 to 50 mm in length, and the thickness of the developer pad may be, for example, in the range of 0.5 to 2 mm.

[0061] (Development Confirmation Zone) Furthermore, the immunochromatographic test piece of the present invention may further comprise a development confirmation zone (development confirmation zone 11 in Figures 4 and 5) in which a substance capable of capturing the label is fixed to the matrix.

[0062] The development confirmation zone according to the present invention functions as a control zone for confirming whether the development of the developer in the immunochromatographic test piece according to the present invention has been successful and determining whether the test is successful. The development confirmation zone according to the present invention is located downstream of the label-containing section. Furthermore, the development confirmation zone according to the present invention is preferably located downstream of the capture body-immobilizing section.

[0063] In the present invention, a "substance capable of capturing the label (hereinafter sometimes referred to as a "control substance")" is a substance capable of binding to the label and capturing the label. Such a control substance is preferably a substance that does not bind to the test substance, and is typically an antibody or antigen capable of binding to the label or detector contained in the label. As such a control substance, for example, an anti-enzyme antibody can be used when the label contained in the label is an enzyme, and an anti-rabbit antibody can be used when the detector contained in the label is a rabbit polyclonal antibody specific to the test substance.

[0064] 4 and 5, the development confirmation zone 11 is formed in a line shape, but the shape of the development confirmation zone in the present invention is not limited to this and can be any shape such as a circle, a polygon, etc. Of these shapes, the shape of the development confirmation zone in the present invention is preferably a line, and when the matrix is ​​in a strip shape, it is more preferably a line shape with a width of 3 to 10 mm and a length of 0.5 to 3 mm.

[0065] The method for immobilizing the control substance in the development confirmation zone according to the present invention is not particularly limited, and any conventionally known method or a method based thereon can be appropriately adopted, including the same methods as those mentioned above as methods for immobilizing the capture substance in the capture substance immobilization section. The amount of the control substance immobilized in the development confirmation zone according to the present invention can be appropriately adjusted depending on the form of the sample, the type of labeling substance or antibody contained in the label, the purpose of detection, etc.

[0066] In the immunochromatographic test strip of the present invention, when the labeled entity (or the labeled entity not captured by the capture entity-immobilizing section, if the development confirmation zone is located downstream of the capture entity-immobilizing section) moves to the development confirmation zone, the labeled entity is captured by the control entity immobilized in the development confirmation zone, and the labeled substance constituting the labeled entity accumulates in the development confirmation zone. As a result, the development confirmation zone exhibits a signal due to the accumulation of the labeled substance, and by detecting this signal, it can be confirmed that the labeled entity has been developed to the development confirmation zone.

[0067] (Absorption Zone) The immunochromatographic test piece of the present invention may further include an absorption zone (absorption zone 9 in Figures 2 to 6) as a member having the function of absorbing the sample and developing solution that have migrated through the matrix, which is a chromatography medium, and the labeled substance that has not been captured in the capture agent immobilization section or development confirmation zone. The absorption zone according to the present invention is preferably located at the most downstream position of the matrix.

[0068] The absorption zone may be integral with the matrix, but is preferably a separate member made of a water-absorbent material, such as the absorbent pad 9 (FIGS. 4 to 6). The substrate for such an absorption zone is not particularly limited, and examples thereof include water-absorbent materials such as cellulose filter paper, nonwoven fabric, cloth, cellulose acetate membrane, synthetic fiber (synthetic fiber such as silica gel, polyethylene terephthalate, polyvinyl alcohol, cellulose), and paper.

[0069] The size of the absorption zone is not particularly limited, but when the matrix is ​​in the form of a strip, for example, the width is in the range of 1 to 10 mm and the length is in the range of 4 to 6 mm, and the thickness of the absorbent pad is in the range of 0.5 to 2 mm.

[0070] (Substrate Zone, Substrate Addition Section) In the immunochromatographic test piece of the present invention, when the labeled substance contained in the label is an enzyme, the substrate may be pre-contained in the developing solution, but it is preferable that the matrix further comprises a substrate zone (substrate zone 10 in Figures 2 to 4) that retains the enzyme substrate in an elutable manner, or an addition section (substrate addition section) to which the enzyme substrate is added during or after development of the developing solution. In this case, it is preferable that the matrix includes the substrate zone or substrate addition section. The substrate zone or substrate addition section according to the present invention is located upstream of the capture body immobilization section. Furthermore, the substrate zone or substrate addition section according to the present invention is preferably located upstream of the label-containing section.

[0071] The method for retaining the substrate in the substrate zone according to the present invention is not particularly limited as long as it allows the substrate to be eluted into the sample or developing solution when developed, and includes methods similar to those listed above as methods for retaining a label in a label-containing zone. The method for adding the substrate to the substrate addition zone according to the present invention is also not particularly limited. The amount of the substrate retained in the substrate zone according to the present invention or the amount of the substrate added to the substrate addition zone can be adjusted appropriately depending on the type of enzyme or substrate, etc.

[0072] Furthermore, the immunochromatographic test strip of the present invention may further include a support (not shown, for example, an adhesive sheet made of plastic, metal, paper, etc.) for supporting the matrix and each pad, as needed. Furthermore, the test strip may further include adhesive tape or the like for adhering and fixing each component together, as long as it does not impair the effects of the present invention.

[0073] The immunochromatographic device of the present invention may be any device as long as it is equipped with the immunochromatographic test piece of the present invention, and may further include, for example, a developer tank (developer tank 12 in Figures 4 to 6) for storing developer upstream of the matrix of the immunochromatographic test piece, a protrusion (protrusion 15 in Figure 6) for supplying the developer in the developer tank to the matrix through the developer pad, and a pusher (pusher 14 in Figures 5 to 6) for applying pressure to the protrusion and moving it, and may also include a case that houses the immunochromatographic test piece of the present invention and is provided with a determination window (determination window 16 in Figure 5) for confirming the detection zone and development confirmation zone.

[0074] In the immunochromatographic test strip of the present invention, the arrangement of each component is as described above and is not particularly limited; however, when the labeling substance contained in the label is an enzyme and the purpose is to perform an enzyme immunoassay, from the viewpoint of excellent accuracy, it is preferable that the immunochromatographic test strip be in the form of a strip, and that the developer supply section, the blood cell removal pad and waterproof layer, and the capture body fixing section be arranged in this order from upstream to downstream, and it is further preferable that the blood cell removal pad be equipped with a label-containing section, and / or that a substrate zone or substrate addition section be arranged between the developer supply section and the blood cell removal pad.

[0075] <Analyte detection method> The present invention also provides a method for detecting a test substance in a sample containing blood cells by immunochromatography, which comprises, in the immunochromatographic test piece of the present invention, a step of adding the sample to the application portion (step 1) and a step of developing a developing solution in the matrix (step 2), and the sample and the developing solution are brought into contact with each other at the protruding portion of the blood cell removal pad.

[0076] As one embodiment of the analyte detection method of the present invention, an analyte detection method (immunochromatography method) using the immunochromatographic test strip shown in FIGS. 1 to 4 will be described below as an example.

[0077] In the analyte detection method of the present invention, first, the sample 13 is added to the addition part 5 by dropping or applying it thereto and allowed to accept it (step 1). It is preferable that the sample 13 is first brought into contact with the label. In this case, the sample 13 may be mixed with the label in advance before being added to the addition part 5, but it is more preferable that the sample 13 is added to the addition part 5 so that it comes into direct contact with the label. For example, it is even more preferable that the addition part 5 also serves as the label-containing part. When the addition part 5 also serves as the label-containing part, contacting the sample 13 with the label held in the label-containing part elutes the label, and, if the analyte is present in the sample 13, an antigen-antibody reaction occurs between the analyte and the detector constituting the label, thereby forming a complex (immune complex) between the analyte and the label (labeling step).

[0078] Furthermore, a developer is supplied to the matrix 1, either simultaneously with or separately from the sample 13 (preferably after the addition of the sample 13), preferably from a developer supply unit 7 upstream of the application unit 5 (step 2). For example, as shown in an immunochromatographic device 202 in FIGS. 5 and 6 , the developer pad 7 can be inserted into the developer tank 12 by applying pressure to the pusher 14 to move the protrusion 15, and the developer can be supplied to the matrix 1 through the developer pad 7. Examples of the developer include a buffer solution adjusted to a pH suitable for the target antigen-antibody reaction. More specific examples of the buffer solution include phosphate buffer, Tris buffer, Good's buffer, borate buffer, and 2-amino-2-methyl-1-propanol (AMP) buffer. The developer may also contain one or more surfactants, blood coagulation inhibitors, and the like.

[0079] Furthermore, when the labeling substance contained in the label is an enzyme, the immunochromatographic test strip of the present invention preferably further comprises a substrate zone 10. When the immunochromatographic test strip further comprises a substrate zone 10, the substrate is eluted into the developing solution as the developing solution passes through the substrate zone 10, and the developing solution containing the substrate flows. The substrate may be added by dropping or applying a substrate solution to the substrate application section simultaneously with or separately from the developing solution. In this case, the solvent for the substrate may be, for example, the buffer solution.

[0080] The developing liquid supplied to matrix 1 moves within matrix 1 in developing direction 4 of the developing liquid due to capillary action. Sample 13 received in application section 5 also moves within blood cell removal pad 2 in developing direction 4 of the developing liquid due to capillary action. At this time, waterproof layer 3 prevents sample 13 from moving vertically from blood cell removal pad 2 to matrix 1.

[0081] After the sample 13 received in the application section 5 moves through the blood cell removal pad 2 in the developing solution development direction 4, blood cells are removed, and the remaining components (e.g., plasma and serum) containing the analyte (preferably a complex of the analyte and a label) come into contact with the matrix 1 at the overhanging section 6 and with the developing solution moving through the matrix 1. The developing solution and the sample (sample 13 from which blood cells have been removed) move through the matrix 1 in the developing solution development direction 4 and, in the embodiment shown in Figures 2 to 6, reach the capture body-immobilizing section 8 (detection zone 8). As a result, the capture body immobilizing section 8 captures the immune complex via the analyte using the capture body immobilizing section 8 (capture step). As a result of the capture of the immune complex by the capture body-immobilizing section 8, the capture body-immobilizing section 8 exhibits coloration or the like due to the labeling substance constituting the label, which can be detected as a signal (detection step) and, if necessary, quantified. Furthermore, when the labeling substance is an enzyme, in the embodiments shown in Figures 2 to 6, a developing solution containing a substrate moves to the capture body immobilizing section 8, and therefore a signal generated by the enzyme-substrate reaction can be detected.

[0082] In the test substance detection method of the present invention, the capture step and the detection step may be carried out simultaneously, or the detection step may be carried out after the capture step. For example, a signal such as coloration that is generated simultaneously with the capture step may be detected with the naked eye, or after capturing the complex in the capture step, a detection step may be carried out in which a treatment appropriate for the labeled substance contained in the complex (addition of a substrate, observation with a fluorescence microscope, detection with a fluorometer, detection with a scintillation counter, etc.) is carried out.

[0083] If the sample does not contain the analyte, the label is not fixed to the capture body-fixing part 8 and moves further downstream, and therefore no signal is detected at the capture body-fixing part 8. Furthermore, if the concentration of the analyte in the sample is low, the amount of label accumulated at the capture body-fixing part 8 decreases, resulting in a weak signal intensity, which makes it possible to quantify the analyte based on the signal intensity.

[0084] In the embodiments shown in FIGS. 4 to 6 , the labeled body (if the development confirmation zone 11 is located downstream of the capture body fixing section 8, the labeled body not captured by the capture body fixing section 8) moves further downstream and reaches the development confirmation zone 11. As a result, the labeled body is captured and detected by the control body fixed in the development confirmation zone 11 (confirmation step). In the embodiments shown in FIGS. 4 to 6 , the confirmation step is performed after the capture step. However, the capture step and the confirmation step may be performed independently or simultaneously at the same location. Alternatively, the capture step may be performed after the confirmation step. As a result of the capture of the labeled body in the development confirmation zone 11, the development confirmation zone 11 exhibits a color or the like due to the labeling substance constituting the labeled body. This color can be detected as a signal, and by quantifying it as necessary, it can be determined whether the sample has migrated normally on the immunochromatographic test strip, i.e., whether the test is successful. The criteria for the determination can be set appropriately depending on the purpose of detection, the amount of test substance, etc. In the embodiment shown in FIGS. 2 to 6, the developing solution is finally absorbed in the downstream absorption zone 9 (absorbent pad 9).

[0085] <Immunochromatography Kit> The immunochromatography kit of the present invention includes at least the immunochromatography test strip of the present invention. The immunochromatography test strip may be included in the immunochromatography device of the present invention.

[0086] Furthermore, the immunochromatography kit of the present invention may further include at least one selected from the group consisting of a sample diluent and a developing solution, as needed. Furthermore, the label, the capture body, the control body, or the antibodies, antigens, labeling substances, etc. that constitute these may also be combined. Furthermore, for example, when the labeling substance is an enzyme, a substrate or a reaction stop solution necessary for detecting the label may also be included. Furthermore, the immunochromatography kit of the present invention may further include instructions for use of the kit.

[0087] The immunochromatographic test strip, immunochromatographic device, test substance detection method, and immunochromatographic kit of the present invention can be used, for example, as research supplies, and can also be used as in vitro diagnostic pharmaceuticals for detecting the test substance that forms the basis for diagnosing a disease related to the test substance.

[0088] The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. In particular, the antibodies, antigens, their amounts, reaction conditions, etc. can be appropriately adjusted.

[0089] (1) Preparation of Immunochromatography Device (Examples 1 to 3) An immunochromatography device having the configuration shown in FIG. 4 was prepared. First, an immunochromatography test strip was prepared. (Matrix, Waterproof Layer) A nitrocellulose membrane (porous membrane, manufactured by Merck or Sartorius) measuring 3.65 mm in width and 50 mm in length was used as matrix 1, with one short side of matrix 1 positioned at the left end. Waterproof tape measuring 3.65 mm in width, 13 mm in length, and 0.025 mm in thickness (polyester film, Scotch tape manufactured by Kuramoto Sangyo Co., Ltd.) was used as waterproof layer 3, with one short side of waterproof layer 3 positioned at the left end. The left end of waterproof layer 3 was attached to matrix 1, aligning it with a position 15 mm from the left end. (Development confirmation zone, detection zone) At a position 12 mm from the right end of matrix 1, 0.54 μL of a control body fluid containing 21.4 μg / mL of anti-ALP antibody was spotted in a line and dried to form development confirmation zone 11. Furthermore, at a position 15 mm from the right end of matrix 1, 0.54 μL of an antibody or antigen solution against the test substance (antigen A or antibody B) was spotted in a line and dried to form detection zone 8 (capturer immobilization section 8). (Substrate zone) At a position 47 mm from the right end of matrix 1, a substrate (5-bromo-4-chloro-3-indolylphosphate (BCIP)) solution was spotted in a line so that the substrate amount was 27.8 μg and dried to form substrate zone 10. (Blood Cell Removal Pad, Addition Portion) With one short side of each porous membrane shown in Table 1 below, measuring 3.65 mm wide and 15.0 mm long, as the left end, 3.04 μL of labeled body fluid containing a label in which an antibody or antigen against a test substance (antigen A or antibody B) was labeled with ALP was spotted and dried on the upper surface at a position 4.75 mm from the left end to form an addition portion 5 and a label-containing portion 5, thereby producing a blood cell removal pad 2 including the addition portion 5 and the label-containing portion 5. The obtained blood cell removal pad 2 was attached to the waterproof layer 3 on the matrix 1, with the left end of the blood cell removal pad 2 aligned with the left end of the waterproof layer 3, using double-sided tape (base material: nonwoven fabric, width 3.65 mm, length 10 mm, manufactured by 3M). This resulted in the blood cell removal pad 2 extending 2.0 mm from the right end of the waterproof layer 3 (waterproof tape), and contacting the matrix 1 only at this protruding portion.

[0090]

[0091] Table 1 shows the thickness, flow rate, water absorption, particle retention capacity, and product name and distributor name of the porous membrane used as the blood cell removal pad in each Example (and the following Comparative Example). In Table 1, "thickness" indicates the thickness (μm) measured with a vernier caliper under atmospheric pressure at a temperature of 20°C and humidity of 50%; "flow rate" indicates the speed (s / 4 cm) at which water passes through the porous membrane from the top to the bottom under atmospheric pressure at a temperature of 20°C and humidity of 50%; and "water absorption" indicates the speed (s / 4 cm) at which water passes through the porous membrane from the top to the bottom under atmospheric pressure at a temperature of 20°C and humidity of 50%. ２ The amount of water absorbed into the porous membrane per unit area (g / cm ２ "Particle retention capacity" indicates the particle diameter (μm) of particles that are retained in the porous membrane by 98% or more, as measured by flow cytometry or the like.

[0092] Next, a developer pad 7 (porous cellulose membrane) and an absorbent pad 9 (synthetic fiber paper made of silica gel, polyethylene terephthalate, polyvinyl alcohol, and cellulose) were attached to the matrix 1 as shown in Figure 4 to prepare an immunochromatographic test strip. The prepared immunochromatographic test strip was fixed in a plastic case having a developer tank 12 filled with developer, a push-in portion 14, a protrusion 15, and a test window 16 as shown in Figures 4 to 6 to prepare an immunochromatographic device.

[0093] (Comparative Examples 1 to 3) The immunochromatography devices of Comparative Examples 1 to 3 were fabricated in the same manner as Examples 1 to 3, except that the waterproof layer 3 (waterproof tape) was not attached to the matrix 1, and the left edge of the blood cell removal pad 2 was aligned 15 mm from the left edge of the matrix 1 so that the blood cell removal pad 2 and the matrix 1 were in direct contact with each other, and the left edge was attached with double-sided tape (substrate: transparent polyester film, width 3.65 mm, length 5 mm, manufactured by Kuramoto Sangyo Co., Ltd.). Example 1 and Comparative Example 1, Example 2 and Comparative Example 2, and Example 3 and Comparative Example 3 all had the same substrate and configuration, except for the presence or absence of a waterproof layer. In the immunochromatography devices obtained in Comparative Examples 1 to 3, the blood cell removal pad 2 was configured so that almost the entire surface thereof was in direct contact with the matrix 1.

[0094] (2) Evaluation of Immunochromatography Device 1: Detection tests were conducted for two types of test substances (antigen A and antibody B) expected to be tested in whole blood samples using the immunochromatography devices prepared in Examples 1 and 3 above. In the immunochromatography devices used in this test, the nitrocellulose membranes used were manufactured by Merck for antigen A and Sartorius for antibody B. First, a pseudo-whole-blood-positive sample was prepared by adding antigen A to a whole blood sample (whole blood sample from a healthy individual) to a concentration of 100 ng / mL. Furthermore, a pseudo-whole-blood-positive sample was prepared by diluting an antibody B-positive plasma sample 20-fold or 200-fold with a whole blood sample (whole blood sample from a healthy individual). Next, each pseudo-whole-blood-positive sample was dropped into the application section 5 of each immunochromatography device. The drop volume was 25 μL in Example 1 and 50 μL in Example 3. Immediately after dropping, the push-in part 14 was pressed to develop the developing solution, and the color development in the test window (detection zone 8 and development confirmation zone 11) was visually confirmed 15 minutes after pressing. Note that, since each pseudo-whole blood positive specimen was used in this test, correct detection indicated a positive result (blue lines were observed in the detection zone 8 and development confirmation zone 11).

[0095] The appearance of the test windows (detection zone 8 and development confirmation zone 11) upon visual confirmation is shown in Figure 7. In Figure 7, the upper line in each test window is the line for development confirmation zone 11 (reference line), and the lower line is the line for detection zone 8 (test line). As shown in Figure 7, in the immunochromatography device (Examples 1 and 3) equipped with the immunochromatography test strip of the present invention, two lines, namely, the blue to light blue lines for detection zone 8 and development confirmation zone 11, could be confirmed, confirming that the test substance could be easily detected from a whole blood sample.

[0096] (3) Evaluation of immunochromatography device 2 A comparison test was conducted on each of the immunochromatography devices prepared in Examples 1 to 3 and Comparative Examples 1 to 3 above, using a whole blood sample (negative sample) from a healthy person as the whole blood sample. In the immunochromatography devices used in this test, all nitrocellulose membranes were manufactured by Merck. In the test, a whole blood sample (negative sample) from a healthy person was used as the whole blood sample for each immunochromatography device, and the amount of blood dropped into the application part 5 was 25 or 30 μL (46 or 55 μL / cm) for Example 1 and Comparative Example 1. ２ ), Example 2 and Comparative Example 2 were 35, 40, or 45 μL (64, 73, or 82 μL / cm ２ ), Example 3 and Comparative Example 3 were 50, 55, or 60 μL (91, 100, or 110 μL / cm ２ ) was used, and the color development in the judgment window (detection zone 8 and development confirmation zone 11) was visually confirmed 15 minutes after pressing down the pressing part 14. However, in Example 3, because development was slow, the confirmation was made 30 minutes after pressing down the pressing part 14. Note that, since negative samples were used as specimens in this test, correct detection indicates a negative result (a blue line (reference line) is observed in the development confirmation zone 11, but no color development is confirmed in the detection zone 8).

[0097] The appearance of the test window (detection zone 8 and development confirmation zone 11) upon visual inspection is shown in Figure 8. In Figure 8, the upper line in each test window is the line of the development confirmation zone 11 (reference line), and the lower line is the line of the detection zone 8 (test line). The appearance of the test window was visually evaluated according to the following criteria: A: The reference line was clearly visible. No coloring other than the reference line was present in the test window. B: More than two-thirds of the reference line was visible. However, unnecessary blue vertical lines were present in the test window, or the reference line color intensity was weak. C: More than two-thirds of the reference line was visible. However, red vertical lines derived from blood were present in the test window. D: Less than two-thirds of the reference line was visible. E: The entire test window was colored red, and the reference line color was difficult to see. A was the highest rating, and a rating of C or higher indicated practical applicability as an immunochromatography device. The evaluation results are also shown in Figure 8.

[0098] As shown in Figure 8, all of the immunochromatographic devices equipped with the immunochromatographic test strip of the present invention (Examples 1 to 3, upper part of Figure 8) were evaluated as C or higher, confirming that the test window could be clearly confirmed even when a whole blood sample was used. On the other hand, all of the immunochromatographic devices equipped with the immunochromatographic test strip without a waterproof layer (Comparative Examples 1 to 3, lower part of Figure 8) were evaluated as D or lower, confirming that the reference line in the test window was unclear or could not be confirmed, and that they are difficult to apply to tests using whole blood samples.

[0099] (4) Investigation of Immunochromatography Devices (4-1) Fabrication of Immunochromatography Devices (Examples 1-1 and 3-1) The position of the label-containing portion was investigated in blood cell removal pads equipped with a label-containing portion. First, in the same manner as in Examples 1 and 3, except that an ALP-labeled antibody against the following antigen C (analyte) was used as the label, one short side of each porous membrane having a width of 3.65 mm and a length of 15.0 mm was set as the left end, and labeled body fluid was spotted and dried on the upper surface at a position 4.75 mm from the left end and in the center of the width direction (within the horizontal line area indicated by a in Figure 9), to form a label-containing portion, thereby fabricating blood cell removal pads 2 including a label-containing portion. Furthermore, using the obtained blood cell removal pad 2, immunochromatography devices of Examples 1-1 and 3-1 were fabricated in the same manner as in Examples 1 and 3, except that an antibody against antigen C was used in the detection zone 8 (capturer immobilization portion 8). The blood cell removal pad 2 was attached with double-sided tape to the waterproof layer 3 on the matrix 1 so that its left edge matched the left edge of the waterproof layer 3. Except for controlling the position of each label-containing portion as described above, the substrate and configuration of each immunochromatography device are the same for Example 1-1 and Example 1, and for Example 3-1 and Example 3. All nitrocellulose membranes used were manufactured by Merck.

[0100] Example 1-2 An immunochromatography device was produced by attaching the obtained blood cell removal pad 2 to the waterproof layer 3 on the matrix 1 in the same manner as in Example 1-1, except that the pad 2 was turned upside down so that the surface on which the labeled body fluid had been spotted and dried was within position c in Figure 9. That is, on the matrix 1, the label-containing portion was arranged so that the label content was greater on the waterproof layer 3 side of the underside of the blood cell removal pad 2. Except for controlling the position of the label-containing portion as described above, the substrate of the immunochromatography device and its configuration were the same as in Examples 1 and 1-1.

[0101] Example 3-2 An immunochromatography device was produced by attaching the obtained blood cell removal pad 2 to the waterproof layer 3 on the matrix 1 in the same manner as in Example 3-1, except that the left and right sides were reversed so that the position of the label-containing portion was within the position b in Figure 9. That is, on the matrix 1, the label-containing portion was located 10.25 mm from the left end of the blood cell removal pad 2, at the center in the width direction (within the grid line portion shown by b in Figure 9). Except for controlling the position of the label-containing portion as described above, the substrate of the immunochromatography device and its configuration were the same as in Examples 3 and 3-1.

[0102] (4-2) Evaluation of Immunochromatography Device 3 Using each immunochromatography device prepared in (4-1) above, a detection test was conducted for a test substance (antigen C) expected to be tested in a whole blood sample. First, a pseudo-positive whole blood sample was prepared by adding antigen C to a whole blood sample (whole blood sample from a healthy individual) to a concentration of 10 ng / mL. Next, the prepared pseudo-positive whole blood sample was dropped onto the application section 5 of each immunochromatography device (on the upper surface of the blood cell removal pad 2, within 5 mm from the left edge, regardless of the position of the label-containing section). The drop volume was 25 μL for Examples 1-1 and 1-2, and 45 μL for Examples 3-1 and 3-2. Immediately after dropping, the push-in section 14 was pressed to develop the developing solution, and the color development in the test window (detection zone 8 and development confirmation zone 11) was visually confirmed 15 minutes after pressing. In this test, a pseudo-positive whole blood sample is used as the sample, and therefore, if the detection is correct, both will show a positive result (blue lines will be observed in the detection zone 8 and the development confirmation zone 11).

[0103] FIG. 10 shows the appearance of the judgment windows (detection zone 8 and development confirmation zone 11) upon visual confirmation. In FIG. 10, the upper line in each judgment window is the line of the development confirmation zone 11 (reference line), and the lower line is the line of the detection zone 8 (test line). The reference line and test line were visually evaluated according to the following criteria: +++: The line color is a blue with a higher saturation than pale blue. ++: The line color is pale blue. +: A pale blue line is observed, but with the minimum color intensity visible in the photograph. -: The test line color is difficult to confirm, or less than two-thirds of the test line is visible. Indeterminable: The reference line color is difficult to confirm, or less than two-thirds of the reference line is visible (test failed). +++ is the highest evaluation, and + or higher indicates practical use as an immunochromatography device. The evaluation results are also shown in FIG. 10.

[0104] As shown in Figure 10, in an immunochromatography device equipped with the immunochromatography test strip of the present invention, the reference line and test line were confirmed with an evaluation of + or higher, regardless of the position of the label-containing portion. In particular, when the label-containing portion in the blood cell removal pad was located at position a in Figure 9, that is, upstream in the direction of development of the developer and overlapping with the application portion where the sample was added, both the reference line and the test line were evaluated highly, and tended to be more preferable.

[0105] As described above, according to the present invention, it is possible to provide an immunochromatographic test piece that can easily detect a test substance in a sample containing blood cells, such as a whole blood specimen, and that can also perform enzyme immunoassays with high accuracy, an immunochromatographic device that includes the test piece, an immunochromatographic kit that includes at least one of these, and a method for detecting a test substance in a sample containing blood cells using at least one of these.

[0106] 101, 102, 103...Immunochromatography test strip, 201, 202...Immunochromatography device, 1...Matrix, 2...Blood cell removal pad, 3...Waterproof layer, 4...Developing direction of developer, 5...Addition section (label-containing section in Examples 1 to 3, 1-1, 3-1), 6...Protruding section, 7...Developer supply section, developer pad, 8...Capturer fixing section (detection zone), 9...Absorption zone, absorption pad, 10...Substrate zone, 11...Development confirmation zone, 12...Developer tank, 13...Sample, 14...Push-in section, 15...Protrusion, 16...Determination window

Claims

1. A test piece for immunochromatography used to detect a test substance in a sample containing blood cells by immunochromatography, comprising: a matrix for developing a developing liquid; a blood cell removal pad including an application area for the sample, laminated on the matrix; and a waterproof layer laminated between the matrix and the blood cell removal pad, wherein the blood cell removal pad has a protruding portion that protrudes beyond the waterproof layer onto the matrix in the direction in which the developing liquid is developed.

2. The immunochromatographic test piece according to claim 1, wherein the length of the protruding portion of the blood cell removal pad is 1 to 4 mm.

3. The immunochromatographic test strip according to claim 1, wherein the blood cell removal pad is a glass fiber membrane.

4. The immunochromatographic test piece according to claim 1, further comprising a capture body fixing section, on the matrix downstream of the blood cell removal pad, to which a capture body capable of capturing a test substance is fixed.

5. An immunochromatographic test piece as described in claim 1, comprising a label-containing portion that retains in an elutable manner the label bound to an antibody or antigen capable of binding to the test substance.

6. The immunochromatographic test piece according to claim 5, wherein the labeling substance is an enzyme.

7. An immunochromatographic test piece as described in claim 6, wherein the developing solution contains a substrate for the enzyme, or the matrix is ​​provided with a substrate zone upstream of the capture body immobilization portion for holding the substrate for the enzyme in an elutable manner, or the matrix is ​​provided with an addition portion for the substrate for the enzyme.

8. The immunochromatographic test piece according to claim 5, wherein a label-containing pad including the label-containing portion is laminated on the blood cell removal pad, or the blood cell removal pad is provided with the label-containing portion.

9. An immunochromatographic device used for detecting a test substance in a sample containing blood cells by immunochromatography, the device comprising an immunochromatographic test piece according to any one of claims 1 to 8.

10. An immunochromatography kit used for detecting a test substance in a sample containing blood cells by immunochromatography, the kit comprising an immunochromatography test piece according to any one of claims 1 to 8.

11. A method for detecting a test substance in a sample containing blood cells by immunochromatography, comprising: a matrix for spreading a developing solution; a blood cell removal pad including an application portion for the sample laminated on the matrix; and a waterproof layer laminated between the matrix and the blood cell removal pad, wherein the blood cell removal pad has an overhanging portion that overhangs onto the matrix in the direction in which the developing solution is spread beyond the waterproof layer, the method for detecting a test substance comprising the steps of: adding the sample to the application portion; and spreading the developing solution on the matrix, and bringing the sample and the developing solution into contact at the overhanging portion of the blood cell removal pad.

Images