JP2012137326A - Specimen analysis tool - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a specimen analysis tool enabling a supplied sample fluid to be efficiently transferred to a detecting part, and more particularly, enabling an efficient transfer to the detecting part even when the amount of the sample fluid is small.SOLUTION: A specimen analysis tool of the present invention comprises: a specimen supplying layer for supplying a specimen; a reagent layer containing a labeling binding substance; a developing layer for developing the specimen; a detecting part where a fixing binding substance is fixed; and a supporting body, in which the developing layer, the reagent layer and the specimen supplying layer are laminated on top of the supporting body whilst the reagent layer is disposed in a state of contacting the developing layer in a flow direction of the specimen in the developing layer, and the specimen supplying layer is disposed such that a downstream lateral face of the specimen supplying layer contacts an upstream lateral face of the reagent layer. The developing layer includes the detecting part, and a water absorption amount per unit volume of the specimen supplying layer is equal to or less than 5.4 mL/cm. According to the specimen analysis tool, a sample fluid supplied to the specimen supplying layer can be efficiently transferred to the detecting part.

Description

  The present invention relates to a sample analysis tool.

  Detection of pathogens such as bacteria and viruses is very important in the diagnosis of infectious diseases. In the diagnosis, since a simple and rapid diagnosis is possible, a specimen analysis tool for detecting the antigen of the pathogen using an immunochromatography method is widely used.

  In the sample analysis tool, for example, a sample supply layer for supplying a sample, a reagent layer having a reagent, and a developing layer for developing the sample are arranged on a support. The reagent layer is disposed adjacent to the upstream side of the development layer, the region on the downstream side of the sample supply layer is stacked on the upstream side of the reagent layer, and the region on the upstream side of the sample supply layer is Laminated on the support. As the reagent, for example, a labeled antibody against the target labeled with colored latex particles or the like is used, and a detection part is formed in the downstream region of the development layer by immobilizing the antibody against the target. (Patent Document 1).

  The measurement principle by the immunochromatography method using the sample analysis tool is as follows. First, a sample liquid containing a specimen is dropped onto the specimen supply layer. The dropped sample liquid moves through the specimen supply layer and reaches the reagent layer. When the sample solution contains the target, the target and the labeled antibody react with each other in the reagent layer to form a complex. Then, the sample solution containing the complex moves through the reagent layer and reaches the spread layer. The sample liquid that has reached the spread layer moves toward the downstream side in the flow direction. In the spreading layer, when the sample solution containing the complex reaches the detection unit, the complex is captured by the immobilized antibody of the detection unit. Since the labeled antibody in the complex is labeled with the colored latex particles, the detection unit is colored by capturing the complex. By detecting the color of the detection unit, the presence or amount of the target in the sample can be analyzed.

  However, the sample analysis tool has the following problems. That is, the above-described sample analysis tool has a problem that sufficient detection sensitivity cannot be obtained because the sample liquid supplied to the sample supply layer does not efficiently move to the detection unit. For this reason, in order to move an amount of the sample sufficiently reacting with the labeled antibody in the reagent layer to the detection unit, it is necessary to add a large amount of the sample solution to the sample supply layer. It was. In order to prepare a large amount of sample solution, it is necessary to dilute the sample with a large amount of extract solution. As a result, a new problem arises that the concentration of the sample in the sample solution is low. . Furthermore, in order to avoid the problem of a decrease in the analyte concentration in the sample solution, the amount of the analyte used for the preparation of the sample solution must be increased. As a result, the required amount of the analyte is reduced. It will increase. Thus, the analysis method becomes complicated by preparing a large amount of sample liquid using a large amount of specimen and supplying a large amount of sample liquid to the specimen analysis tool.

JP 2010-60297 A

  Accordingly, the present invention can efficiently move the supplied sample liquid to the detection unit, for example, more specifically, even when the amount of the sample liquid is small, the sample liquid is efficiently moved to the detection unit. It is an object of the present invention to provide a sample analysis tool that can be used.

The sample analysis tool of the present invention comprises:
A sample supply layer for supplying a sample, a reagent layer containing a labeled binding substance, a development layer for developing the specimen, a detection unit on which an immobilized binding substance is immobilized, and a support,
The spread layer, the reagent layer, and the specimen supply layer are laminated on the support,
In the flow direction of the specimen in the spreading layer,
The reagent layer is disposed in contact with the spreading layer,
The sample supply layer is disposed in a state where the downstream side surface of the sample supply layer is in contact with the upstream side surface of the reagent layer,
The spread layer includes the detection unit,
A water absorption amount per unit volume of the specimen supply layer is 5.4 mL / cm ³ or less.

  In the sample analysis tool of the present invention, as described above, the sample supply layer is disposed in a state where the side surface on the downstream side of the sample supply layer is in contact with the side surface on the upstream side of the reagent layer, and the condition A specimen supply layer that satisfies With such a configuration, the supplied sample liquid can be efficiently moved to the detection unit. Thus, since the sample liquid can be efficiently moved, for example, the amount of the sample liquid can be reduced and the amount of the specimen used for preparing the sample liquid can be reduced. Therefore, according to the sample analysis tool of the present invention, even a small amount of a sample derived from a living body can be efficiently moved to the detection unit with a small amount of the sample liquid, and detection with excellent sensitivity can be realized. It is.

FIG. 1A is a cross-sectional view showing an example of the sample analysis tool of the present invention, and FIG. 1B is a cross-sectional view showing another example of the sample analysis tool of the present invention. 2A is a cross-sectional view showing an example of the sample analysis tool of the present invention, and FIG. 2B is a cross-sectional view showing another example of the sample analysis tool of the present invention. FIG. 3 is a cross-sectional view showing an example of the sample analysis tool of the present invention. FIG. 4 is a cross-sectional view showing an example of the sample analysis tool of the present invention. FIG. 5 is a cross-sectional view showing an example of a conventional sample analysis tool.

In the sample analysis tool of the present invention, as described above, a sample supply layer for supplying a sample, a reagent layer containing a labeled binding substance, a development layer for developing the specimen, and an immobilized binding substance are immobilized. Including a detection unit and a support,
The spread layer, the reagent layer, and the specimen supply layer are laminated on the support,
In the flow direction of the specimen in the spreading layer,
The reagent layer is disposed in contact with the spreading layer,
The sample supply layer is disposed in a state where the downstream side surface of the sample supply layer is in contact with the upstream side surface of the reagent layer,
The spread layer includes the detection unit,
A water absorption amount per unit volume of the specimen supply layer is 5.4 mL / cm ³ or less.

  In the present invention, the water absorption amount per unit volume of the specimen supply layer means the maximum amount of water that the specimen supply layer can hold.

The water absorption can be determined by the following method. First, a specimen supply layer having an external shape of 30 cm wide × 1 cm long × thickness 0.014 cm (overall volume including voids 30 × 1 × 0.014 = 0.42 cm ³ ) is prepared, Put on the table. 0.1 mL of the test solution is dropped into the center of the upper surface of the specimen supply layer. The test solution is 17.5 mmol / L Tris (pH 8.4) containing 70 mmol / L NaCl and 0.7 w / v% BSA. Then, the amount of liquid immediately before the test liquid overflows from the specimen supply layer is divided by a volume of 0.42 cm ³ , and this calculated value is taken as the water absorption amount per unit volume. This method is a method for determining the amount of water absorption per unit volume, which is a condition of the specimen supply layer in the present invention, and the present invention is not limited by this method.

As described above, the water absorption amount per unit volume of the specimen supply layer is 5.4 mL / cm ³ or less, and the range thereof is, for example, 4.5 to 5.4 mL / cm ³ , preferably 5 24 mL / cm ³ .

  The material of the specimen supply layer is not particularly limited, and examples thereof include glass fiber and cellulose fiber. Examples of the form of the specimen supply layer include a nonwoven fabric.

  As the specimen supply layer, for example, a commercially available product may be used, and examples thereof include a glass fiber pad (trade name GFCP001000 G041 Glass Fiber Conjugate Pad) manufactured by Millipore.

The size of the specimen supply layer is not particularly limited, and can be appropriately determined according to the amount of the sample liquid containing the specimen supplied to the specimen supply layer. As described above, the sample analysis tool of the present invention can be efficiently moved to the spreading layer even if the amount of the sample solution is small. For this reason, for example, when the amount of the sample liquid containing the specimen is about 50 μL, the area of the specimen supply layer is preferably 0.1 to 1.5 cm ² , more preferably 0.3 to ¹ cm ² . .

  In the sample analysis tool of the present invention, for example, when a sample solution containing the sample is supplied to the sample supply layer, the sample is introduced into the reagent layer, and further introduced from the reagent layer into the development layer, The specimen moves in the development layer in the downstream direction. Thereby, a complex of the labeled binding substance and the target in the sample is formed in the reagent layer, and the reached complex is captured by the immobilized binding substance in the detection unit. The target can be analyzed by detecting the label of the labeled binding substance in the complex captured by the immobilized binding substance in the detection unit.

  The labeled binding substance is a binding substance labeled with the label. The binding substance of the labeled binding substance is not particularly limited as long as it can bind to the target. Examples of the binding substance include antibodies, antigens and nucleic acids.

When the target is an antigen, for example, the labeled binding substance is preferably a labeled antibody against the antigen. When the binding substance of the labeled binding substance is an antibody, the type thereof is not particularly limited, and examples thereof include immunoglobulin molecules such as IgG, IgA, IgM, IgD, IgE and IgY, and Fab, Fab ′, Examples thereof include antibody fragments (also referred to as antigen-binding fragments) such as F (ab ′) ₂ . The antibody may be derived from, for example, humans or animal species such as non-human mammals such as mice, rabbits, cows, pigs, horses, sheep and goats, and birds such as chickens. The antibody may be prepared, for example, from serum derived from the animal species by a conventionally known method, or a commercially available antibody may be used. The antibody may be, for example, a polyclonal antibody or a monoclonal antibody.

  When the target is an antibody, for example, the labeled binding substance is preferably a labeled antibody against the antibody. When the binding substance of the labeled binding substance is an antibody, the type thereof is not particularly limited and is the same as described above, and examples thereof include an antibody that binds to the Fc region of the target antibody and an antibody fragment thereof. .

  The label of the labeled binding substance is not particularly limited as long as it can be detected. Examples of the label include colored insoluble carrier particles or enzymes.

  The colored insoluble carrier particles are not particularly limited. For example, colored latex particles, metal colloid particles, colored polymethyl methacrylate particles, colored polylactic acid particles, colored porous glass particles, colored silica particles, colored agarose particles, colored dextran particles. Etc. The colored latex particles are not particularly limited, and examples thereof include blue latex particles and red latex particles. The metal colloid particles are not particularly limited, and examples thereof include gold colloid particles and platinum colloid particles. The average particle diameter of the colored insoluble carrier particles is not particularly limited. In the case of the colored latex particles, the average particle diameter is, for example, in the range of 0.05 μm to 5 μm, and preferably in the range of 0.1 μm to 1 μm. In the case of the metal colloid particles, the average particle diameter is, for example, in the range of 2 nm to 100 nm, and preferably in the range of 10 nm to 50 nm.

  When the label is the colored insoluble carrier particles, for example, the method for preparing the labeled binding substance is not particularly limited. As a specific example, for example, the colored insoluble carrier particles are suspended in a solvent, and the binding substance such as the antibody is added to the suspension. By reacting the colored insoluble carrier particles and the binding substance in the reaction solution, the binding substance labeled with the colored insoluble carrier particles can be prepared. The solvent is not particularly limited, and examples thereof include water and a buffer solution. The buffer is not particularly limited, and examples thereof include Tris buffer, phosphate buffer, acetate buffer, and borate buffer. The pH of the buffer solution is not particularly limited, and is, for example, in the range of pH 4 to 10, and preferably in the range of pH 6 to 9. In the suspension, the concentration of the colored insoluble carrier particles is not particularly limited, and is, for example, in the range of 0.001 to 10% by weight, and preferably in the range of 0.01 to 1% by weight. In the reaction solution, the concentration of the binding substance such as the antibody is not particularly limited, and is, for example, in the range of 0.01 to 20 mg / mL, preferably in the range of 0.1 to 5 mg / mL. is there.

  The enzyme is not particularly limited, and examples thereof include those that cause coloration, color development, or luminescence upon reaction with a substrate. Examples of the enzyme include peroxidase, alkaline phosphatase, β-D-galactosidase and the like.

  The substrate is not particularly limited, and can be determined as appropriate depending on, for example, the type of the enzyme. Examples of the substrate include 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3 ′, 5,5′-tetramethylbenzidine (TMB), diaminobench Gin (DAB), 5-bromo-4-chloro-3-indolylphosphoric acid (BCIP), 4-methylumbelliferyl-β-D-galactoside (4MUG), 3- (2′-spiroadamantane) -4 -Methoxy-4- (3 ″ -β-D-galactopyranosyl) phenyl-1,2-dioxetane (AMGPD) and the like.

  When the label is the enzyme, for example, the method for preparing the labeled binding substance is not particularly limited, and for example, a conventionally known method can be adopted.

  The immobilized binding substance is a binding substance immobilized on the detection unit of the development layer. The binding substance of the immobilized binding substance is not particularly limited as long as it can bind to the target. Examples of the binding substance include antibodies, antigens and nucleic acids.

  When the target is an antigen, for example, the immobilized binding substance is preferably an antibody against the antigen. When the binding substance of the immobilized binding substance is an antibody, the type thereof is not particularly limited, and includes the immunoglobulin molecule and antibody fragments thereof as described above.

  When the target is an antibody, for example, the immobilized binding substance is preferably an immobilized antigen for the antibody.

  The method for immobilizing the immobilized binding substance on the spreading layer is not particularly limited, and a conventionally known method can be adopted. As a specific example, for example, using a coating apparatus or the like, the coating solution containing the immobilized binding substance can be applied to a predetermined region of the spreading layer and dried. And the said predetermined area | region becomes the said detection part in the said expansion | deployment layer. The coating solution is not particularly limited, and can be prepared, for example, by suspending the immobilized binding substance such as the immobilized antibody or the immobilized antigen in a solvent. The solvent is not particularly limited, and examples thereof include water and a buffer solution. The buffer solution is not particularly limited, and examples thereof include the aforementioned buffer solution. Further, for example, the concentration of the immobilized binding substance in the coating solution is not particularly limited, and is, for example, in the range of 0.01 to 20 mg / mL, preferably in the range of 0.1 to 5 mg / mL. is there. The drying conditions are not particularly limited, and may be air-dried using, for example, a dryer.

  The types of the labeled binding substance and the immobilized binding substance may be the same or different, for example. It is preferable that the labeled binding substance and the immobilized binding substance bind to different sites of the target, for example. When the target is an antigen, it is preferable that the labeled binding substance and the immobilized binding substance have, for example, different sites on the target as epitopes. The combination of the target, the labeled binding substance and the immobilized binding substance is not particularly limited. As a specific example, when the target is an antigen, for example, the labeled binding substance and the immobilized binding substance are each preferably an antibody against the antigen, and when the target is an antibody, for example, the fixation It is preferable that the labeled binding substance is an antigen against the antibody, and the labeled binding substance is an antibody against the antibody.

  The support is preferably, for example, a liquid that cannot move from the surface to the inside. For example, the surface of the support is preferably liquid non-permeable, which does not allow liquid penetration, and is preferably hydrophobic.

  In the present invention, the specimen is not limited at all. Examples of the specimen include nasal aspirate, nasal wash, nasal rinse, nasal discharge, throat swab, gargle, saliva, whole blood, serum, plasma, sweat, urine and other biological specimens, foods, beverages, etc. Samples derived from food and drink, environmental samples such as seawater, river water, and domestic wastewater. For example, the specimen may be liquid or solid. Examples of the sample solution supplied to the sample analysis tool of the present invention include the liquid sample itself, a diluted solution obtained by suspending, dispersing, or dissolving these samples in a solvent. The sample solution may be, for example, a solid sample suspended in, dispersed or dissolved in the solvent, a sample extracted with the solvent, or a diluted solution thereof. The solid sample is not particularly limited, and examples thereof include biological samples such as cells and stool, foods such as animals and plants, and food samples such as processed foods. The solvent is not particularly limited, and examples thereof include water and a buffer solution. The buffer solution is not particularly limited, and examples thereof include the aforementioned buffer solution. The solvent may further contain, for example, a surfactant, an antibacterial agent and the like. The surfactant is not particularly limited, and examples thereof include an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. The antibacterial agent is not particularly limited, and examples thereof include sodium azide, 5-chloro-2-methyl-4-isothiazolin-3-one, and 2-methyl-4-isothiazolin-3-one.

  In the sample analysis tool of the present invention, as the sample liquid containing the sample, for example, the liquid sample is preferably supplied as it is or the diluent is supplied. As described above, the sample analysis tool of the present invention can be efficiently moved even with a small amount of sample liquid. For this reason, when using the said dilution liquid as the said sample liquid, it is preferable to use the said dilution liquid diluted with the low dilution rate, for example. According to the sample analysis tool of the present invention, even such a sample solution can be efficiently developed. The low dilution rate is not particularly limited, and is, for example, 8 times or less, preferably 4 times or less.

  The target to be analyzed by the sample analysis tool of the present invention is not particularly limited. Examples of the target include influenza A virus, influenza B virus, influenza C virus, adenovirus, RS virus, coronavirus, astrovirus, norovirus, measles virus, rotavirus, human immunodeficiency virus (HIV), human T cell leukemia virus (HTLV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes virus, mycoplasma, syphilis treponema, chlamydia trachomatis, tuberculosis, coliform group, group A streptococcus, group B Streptococcus, Streptococcus pneumoniae, Staphylococcus, MRSA, Legionella, Enterohemorrhagic Escherichia coli O157, Verotoxin, Salmonella, Clostridium difficile, Helicobacter pylori, CRP, HBs antigen, HBs antibody, HBc antigen, HBc anti , HBe antigen, HBe antibody, prostate specific antigen (PSA), human chorionic gonadotropin (hCG), luteinizing hormone (LH), troponin T, troponin I, myoglobin, D-dimer, fecal hemoglobin, hemoglobin A1c, Examples include pathogen antigens such as IgE antibodies, biological components such as antibodies, cancer markers, hormones, residual agricultural chemicals, environmental hormones, and allergens in foods. These are examples, and the present invention is not limited to these.

  The sample analysis tool of the present invention may further include, for example, a developing solution supply unit. By the developing solution supply means, for example, a developing solution can be further supplied to a desired part of the sample analysis tool. Thus, by supplying the developing solution, the specimen can be moved more efficiently. The developing solution supply means is preferably arranged so that the developing solution can be supplied to, for example, a sample supply layer, a reagent part, or a developing layer.

  The sample analysis tool of the present invention can be used by being set in an analyzer, for example. The analyzer includes, for example, an analysis unit that analyzes a state of the arrangement unit of the sample analysis tool and the detection unit of the sample analysis tool. According to the analyzer, for example, the sample can be analyzed by setting the sample analysis tool in the arrangement unit and analyzing the state of the detection unit of the sample analysis tool by the analysis unit. The analysis unit includes, for example, an irradiation unit that irradiates light to the detection unit of the sample analysis tool, and a detection unit that detects reflected light from the detection unit or transmitted light transmitted through the detection unit. As described above, when the complex is held by the detection unit, for example, the detection unit is colored. Therefore, the presence or amount of the target in the sample can be determined by irradiating the detection unit with light and detecting the reflected light or transmitted light. The analyzer may further include, for example, a developing solution supply unit. The developing solution supply means is not particularly limited, and may be any means that can supply the developing solution to the sample analysis tool set in the analyzer, for example. The developing solution supply means includes, for example, draining means, and the developing solution can be supplied to the sample analysis tool by the draining means. The drainage unit may be, for example, an intake / exhaust unit. In this case, the developing solution can be supplied to the sample analysis tool by sucking the developing solution and subsequently discharging the developing solution by the sucking / extracting unit. .

  In the sample analysis tool, the sample supply layer, the reagent layer, and the development layer are laminated on the support, and the sample supply layer, the reagent layer are arranged from the upstream side in the flow direction of the sample. And the spreading layer is disposed.

  As described above, the spreading layer and the reagent layer may be arranged so that they are in contact with each other. The development layer and the reagent layer may be in a state where the downstream end of the reagent layer is in contact with the upstream end of the development layer (adjacent state) in the flow direction, for example. The form of the contact is not particularly limited. For example, it is preferable that side surfaces of the end portions in the flow direction are in contact with each other. The spreading layer and the reagent layer are, for example, such that, in the flow direction, the downstream end of the reagent layer is stacked on the upstream end of the spreading layer, and the other region of the reagent layer However, it may be laminated on the support. In such a stacked state, for example, development in the thickness direction occurs in addition to the flow direction, so that the sample liquid can be developed more efficiently.

  As described above, the reagent supply layer and the sample supply layer are arranged such that the side surface on the downstream side of the sample supply layer is in contact with the side surface on the upstream side of the reagent layer.

  In the sample analysis tool of the present invention, the member disposed on the support may be disposed directly on the surface of the support, for example, or disposed on the support via another component member. May be. The other constituent member is preferably, for example, a liquid that cannot move from the surface to the inside. For example, the surface of the constituent member is preferably liquid non-permeable, which does not allow liquid to penetrate, and is preferably hydrophobic.

  Next, the sample analysis tool of the present invention will be described with examples. The present invention is not limited to the following examples.

  The sample analysis tool of this example is a form in which the sample supply layer, the reagent layer, and the development layer are laminated on the support described above. Unless otherwise indicated, the embodiment described in this embodiment can be applied to each sample analysis tool.

  The sample analysis tool is shown in FIG. FIG. 1A is a cross-sectional view of the sample analysis tool 101 of this example in the sample flow direction. The sample analysis tool 101 includes a support 11, a development layer 12, a sample supply layer 13, and a reagent layer 14. The sample supply layer 13, the reagent layer 14, and the development are provided on the support 11 from the upstream in the sample flow direction. Layers 12 are arranged in this order. The downstream end of the sample supply layer 13 is in contact with the upstream end of the reagent layer 14, and the downstream end of the reagent layer 14 is in contact with the upstream end of the development layer 12. ing. The development layer 12 has a detection unit 15 on which an immobilized antibody is immobilized.

  The support 11 is not particularly limited, and as described above, the surface thereof is preferably liquid non-permeable and preferably hydrophobic. The support 11 may be, for example, a base material whose surface has been subjected to a hydrophobic treatment, or a base material formed from a hydrophobic material. The hydrophobic material is not particularly limited, and examples thereof include polyethylene terephthalate, polyethylene, polystyrene, and polyester. For example, the surface of the support 11 preferably has a non-porous structure. In this case, the support 11 may be, for example, a base material having a non-porous layer laminated on the surface or a base material made of a non-porous body. The shape of the support 11 is not particularly limited, and examples thereof include a film shape, a sheet shape, and a plate shape. The shape and size of the support 11 are not particularly limited, and can be appropriately set according to the configuration of the sample analysis tool.

  The development layer 12 is not particularly limited, and may be, for example, a liquid that can move inside and / or on the surface thereof. For example, the spreading layer 12 preferably exhibits a capillary action, and includes a porous body. Specific examples include cellulose membranes, cellulose derivative membranes such as cellulose acetate and nitrocellulose, and porous materials such as glass filters and filter paper. The shape of the spreading | development layer 12 is not specifically limited, For example, a rectangle etc. are mention | raise | lifted. The size of the spreading layer 12 is not particularly limited and can be set as appropriate. The shape of the development layer 12 is not particularly limited and can be set as appropriate.

  The specimen supply layer 13 only needs to satisfy the above conditions, and the material thereof is not particularly limited. The shape and size of the specimen supply layer 13 are not particularly limited and can be set as appropriate.

  The material of the reagent layer 14 is not particularly limited. The reagent layer 14 is preferably capable of passing a liquid, for example. Examples of the base material of the reagent layer 14 include a porous body. Examples of the material of the reagent layer 14 include polyethylene, glass fiber, rayon, nylon, paper, and cellulose. The shape and size of the reagent layer 14 are not particularly limited and can be set as appropriate.

  The reagent layer 14 has the labeled antibody as described above. The reagent layer 14 can be produced, for example, by impregnating the substrate with an antibody solution containing the labeled antibody and then drying. In the antibody solution, the concentration of the labeled antibody is not particularly limited, and is as described above, for example.

  As described above, the detection unit 15 is formed by immobilizing an immobilized antibody on the development layer 12.

  The development layer 12, the specimen supply layer 13, and the reagent layer 14 can be arranged on the support 11 by, for example, a conventional method. Specifically, for example, the support 11 may be fixed using a double-sided tape or an adhesive.

  The size of the sample analysis tool 101 is not particularly limited, and can be exemplified as follows, for example. Hereinafter, for the sample analysis tool and each member constituting it, the size in the flow direction of the sample is referred to as `` length '', and the size in the direction perpendicular to the flow direction is referred to as `` width '' on the surface of each member, The size in the direction perpendicular to the length and the width is referred to as “thickness” (hereinafter the same). The overall size of the sample analysis tool 101 is, for example, a length of 56 to 84 mm and a width of 3 to 5 mm. The size of the support 11 is, for example, a length of 56 to 84 mm and a width of 3 to 5 mm. The size of the spreading layer 12 is, for example, a length of 16.8 to 25.2 mm and a width of 3 to 5 mm. The size of the sample supply layer 13 is, for example, a length of 2.5 to 12 mm and a width of 3 to 5 mm. The size of the reagent layer 14 is, for example, 8 to 12 mm in length and 3 to 5 mm in width. The amount of the sample solution supplied to the sample analysis tool 101 is not particularly limited, and can be reduced as compared with the conventional case as described above. For example, the amount is 20 to 100 μL, and preferably 30 to 60 μL. In the sample solution, the dilution rate of the specimen is not particularly limited and can be appropriately determined according to the type of the specimen. For example, when the specimen is a nasal discharge-derived specimen, it is 2 to 7 times, preferably 3 to 5 times.

  Next, an example of a sample analysis method using the sample analysis tool 101 of this example will be described with reference to FIG.

  First, the sample solution is dropped on the specimen supply layer 13. The dropped sample liquid moves in the specimen supply layer 13 in the downstream direction. In the sample analysis tool 101 of this example, as described above, the sample supply layer 13 is disposed on the support 11 from which the liquid cannot move from the surface to the inside. Therefore, the sample liquid moves in the downstream direction (the arrow direction in the drawing) in the sample supply layer 13 without moving to the upstream side of the sample supply layer 13. When the sample solution reaches the reagent layer 14 disposed on the downstream side of the specimen supply layer 13, the sample solution further moves in the reagent layer 14 in the downstream direction. At this time, the labeled antibody in the reagent layer 14 is mixed into the sample solution by the sample solution. When the target antigen is contained in the sample solution, the antigen and the labeled antibody in the reagent layer 14 are combined by an antigen-antibody reaction to form a complex of the antigen and the labeled antibody. The The sample solution containing the complex passes through the reagent layer 14 and reaches the development layer 12. When the sample solution reaches the detection unit 15, the complex is captured by the immobilized antibody of the detection unit 15. Specifically, the immobilized antibody and the antigen in the complex are bound to form a complex of the immobilized antibody, the antigen and the labeled antibody. For example, when an antibody labeled with the aforementioned colored insoluble particles is used as the labeled antibody, the detection unit 15 is colored by the formation of the complex. By detecting the coloring of the detection unit 15, the presence or amount of the target in the sample can be analyzed. The detection of the coloring may be performed, for example, visually, or may be optically measured by reflectance, transmittance, or the like using an optical analyzer or the like.

  The conditions for the sample analysis method are not particularly limited, and for example, it is preferably performed under conditions of 10 to 40 ° C.

  In the sample analysis method, for example, a developing solution may be further added. Thus, by supplying the developing solution, the specimen can be moved more efficiently. The developing solution addition site is, for example, the specimen supply layer 13, the reagent layer 14, or the development layer 12, and preferably the specimen supply layer 13. The timing of adding the developing solution is not particularly limited, and is preferably after the sample solution is added, and specifically, for example, within 2 minutes from the addition of the sample solution. The developing solution is not particularly limited, and examples thereof include water and a solvent such as a buffer solution.

  An example in which the reagent layer has a different form with respect to the sample analysis tool of this example is shown in FIG. FIG. 1B shows a cross-sectional view of the sample analysis tool 102 of this example in the flow direction. 1B, the same portions as those in FIG. 1A are denoted by the same reference numerals. As shown in FIG. 1B, in the sample analysis tool 102, the reagent layer 24 has its downstream end laminated on the development layer 12, and the remaining region is arranged on the support 11. For example, the remaining region may be in contact with the upstream end of the development layer 12.

  FIG. 2 shows an example in which the positional relationship between the support and the sample supply layer is different in the sample analysis tool of this example. In FIG. 2, the same parts as those in FIG. FIG. 2A shows a cross-sectional view of the sample analysis tool 104 of this example in the flow direction. As shown in FIG. 2A, the sample analysis tool 104 is placed on the support 11 so that the upstream end of the support 11 and the upstream end of the sample supply layer 13 are at the same position. The specimen supply layer 13 is disposed. FIG. 2B shows a cross-sectional view of the sample analysis tool 105 of this example in the flow direction. As shown in FIG. 2B, the sample analysis tool 105 is placed on the support 11 in a state where the upstream end of the support 11 is downstream of the upstream end of the sample supply layer 13. A specimen supply layer 13 is disposed. In the sample analysis tool of FIG. 1B, the support and the sample supply layer may be in the positional relationship shown in FIGS. 2A and 2B.

  The sample analysis tool of this example may further include an absorption layer, for example. As described above, the sample analysis tool further includes the absorption layer 16, so that the liquid can be more efficiently moved in the development layer 12. An example of a sample analysis tool provided with an absorption layer is shown in FIG. 3, the same parts as those in FIG. 1 are denoted by the same reference numerals. FIG. 3 shows a cross-sectional view of the sample analysis tool of this example in the flow direction. The sample analysis tool 106 in FIG. 3 has a configuration in which the sample analysis tool 101 in FIG. In the sample analysis tool 106, the absorption layer 16 is disposed on the support 11, and the upstream end of the absorption layer 16 is in contact with the downstream end of the development layer 12. For example, as shown in FIG. 3, the upstream end of the absorbent layer 16 may be laminated on the downstream end of the development layer 12, and the remaining region may be disposed on the support 11. For example, the remaining region may be in contact with the downstream end of the development layer 12. Also for the sample analysis tools of FIGS. 1B, 2A, and 2B, an absorption layer may be disposed as shown in FIG. The sample analysis tool 103 in FIG. 4 shows a form in which the absorption layer 16 is arranged on the sample analysis tool in FIG.

  The absorbent layer 16 is not particularly limited and is preferably capable of absorbing a liquid, for example, a porous body. The material of the porous body is not particularly limited, and examples thereof include the same materials as the reagent layer or the development layer described above. The shape and size of the absorbent layer are not particularly limited and can be set as appropriate. The size of the absorption layer 16 is, for example, a length of 28 to 32 mm, a width of 3 to 5 mm, and a thickness of 1434 to 1467 μm.

The target analysis method of the present invention uses the sample analysis tool of the present invention, supplies a sample solution to the sample supply layer of the sample analysis tool, and causes the sample solution to develop on the development layer. Forming a complex of the target in the sample solution and the labeled binding substance of the reagent layer, and capturing the complex by the immobilized binding substance in the detection unit;
And a step of detecting the label of the labeled binding substance in the captured complex.

  The target analysis method of the present invention is characterized by using the sample analysis tool of the present invention, and other processes and conditions are not limited at all. The target analysis method of the present invention can be performed, for example, in the same manner as described in the sample analysis tool of the present invention.

  Next, examples of the present invention will be described. The present invention is not limited to the following examples.

[Example 1]
In this example, influenza A virus (FluA) in the sample solution was detected using the sample analysis tool 106 shown in FIG.

  Hereinafter, for the sample analysis tool and each member constituting it, the size in the flow direction of the sample is referred to as `` length '', and the size in the direction perpendicular to the flow direction is referred to as `` width '' on the surface of each member, The size in the direction perpendicular to the length and the width is referred to as “thickness” (hereinafter the same).

(1) Preparation of specimen analysis tool (1-1) Preparation of blue latex-labeled anti-FluA antibody solution Blue latex particles (particle size 0.3 μm, manufactured by Ceradyne) were added to 20 mmol / L borate buffer solution (pH 8.2). ) Was suspended to a concentration of 1% by weight to prepare a suspension. The suspension was mixed with an anti-FluA monoclonal antibody (manufactured by Fitzgerald) at a concentration of 0.4 mg / mL at a volume ratio of 1: 1 and allowed to react at room temperature for 60 minutes. Next, after centrifuging the reaction solution, the supernatant was removed and resuspended in a borate buffer solution (pH 8.2) containing 1 w / v% bovine serum albumin. The resuspension was centrifuged and the supernatant was removed and suspended in 2.5 mL of Tris buffer. This was used as a blue latex labeled anti-FluA antibody solution.

(1-2) Spreading layer forming sheet A porous nitrocellulose membrane (manufactured by Millipore) was cut into a strip shape. The cut membrane is set in an antibody coating device (manufactured by Biodot), and an anti-FluA monoclonal antibody (manufactured by Fitzgerald) is applied in a line shape to a part away from one end of the long side of the band, and detected. Part was formed. The membrane coated with the antibody was dried. Thus, a development layer forming sheet in which the antibody was immobilized on the membrane was produced.

(1-3) Reagent Layer Forming Sheet 2.2 mL of the blue latex-labeled anti-FluA antibody solution was impregnated into a glass filter (Millipore, trade name GFCP001000) and dried. This produced the reagent layer forming sheet.

(1-4) Specimen Supply Layer Forming Sheet Glass fiber (trade name GFCP001000 G041 Glass Fiber Conjugate Pad, manufactured by Millipore) was used as the specimen supply layer forming sheet. The glass fiber has a water absorption per unit volume of 5.24 mL / cm ³ , a weight of 72-78 g / m ² , a thickness of 0.35 to 0.51 mm, a tensile MD> 150 N / 50 mm, a tensile CD of 80 N / 50 mm, The air permeability was 2300-3300 L / m ² / sec.

(1-5) Absorbing layer forming sheet Filter paper (manufactured by Whatman) was used as the absorbing layer forming sheet.

(1-6) Support Forming Sheet A plastic sheet with a double-sided tape (trade name Sony Chemical T-4100) was used as the support forming sheet.

(1-7) Preparation of specimen analysis tool First, the spread layer forming sheet was laminated on the center region in the width direction of the support forming sheet. At this time, in the spread layer forming sheet, the region side where the detection portion was formed was set as the downstream side in the specimen flow direction. Next, the absorbent layer forming sheet was laminated on the downstream end portion of the support forming sheet from the downstream end portion of the spreading layer forming sheet. At this time, the absorbent layer forming sheet was disposed so as to overlap the developing layer forming sheet by 7 mm. Then, the reagent layer forming sheet was laminated on the support forming sheet on the upstream side of the developing layer forming sheet so as to be in contact with the developing layer forming sheet. Subsequently, the specimen supply layer forming sheet was laminated on the support forming sheet on the upstream side of the reagent layer forming sheet so as to be in contact with the reagent layer forming sheet. In addition, each said sheet | seat was laminated | stacked on the said sheet | seat for support body formation with the double-sided tape. The sheet thus laminated was cut into a strip shape having a width of 4 mm along the flow direction of the sample, and a sample analysis tool 106 having a width of 4 mm and a total length of 77 mm was produced. In the sample analysis tool 106, the cutting portion of the developing layer forming sheet is the developing layer 12, the cutting portion of the reagent layer forming sheet is the reagent layer 114, and the cutting portion of the sample supplying layer forming sheet Is the specimen supply layer 13, and the cut portion of the absorbent layer forming sheet is the absorbent layer 16.

  The sample analysis tool 106 had an overall length of 77 mm and an overall width of 4 mm. The support 11 has a length of 77 mm and a width of 4 mm, the development layer 12 has a length of 21 mm and a width of 4 mm, the specimen supply layer 13 has a length of 10 mm, a width of 4 mm, a thickness of 0.35 to 0.51 mm, and the reagent layer 14 has The length was 10 mm, the width was 4 mm, and the absorbent layer 16 was 29 mm long and 4 mm wide. Further, in the spread layer 12, the length from the upstream end to the upstream end of the detection unit 15 was 7 mm.

(2) Detection of influenza virus Using the sample analysis tool 106 of this example, the influenza virus in the sample was analyzed as follows. In this example, A / Aichi / 2/68 (H3N2) was used as the specimen.

  First, 100 μL of the specimen was suspended in an extract (Tris buffer) to prepare 400 μL of a suspension. The suspension was used as a sample solution. 50 μL of the sample solution was dropped on the specimen supply layer 13. Within 2 minutes immediately after the dropping of the sample, 120 μL of the extract was added to the sample supply layer 13 as a developing solution. Then, 15 minutes after the dropping of the specimen, the color of the detection unit 15 was visually observed (n = 2). Further, as a control, the extract was dropped on the specimen supply layer 13 and similarly observed visually (n = 2).

(Comparative Example 1)
The same sheet as in Example 1 was used, except that a nitrocellulose membrane (Millipore) was used instead of the glass fiber for the preparation of the specimen supply layer forming sheet, as shown below. Thus, the sample analysis tool shown in FIG. 5 was produced. FIG. 5 is a cross-sectional view of the sample analysis tool 107 of Comparative Example 1 in the sample flow direction.

  The spread layer forming sheet was affixed on the center region in the width direction of the support forming sheet. At this time, in the spread layer forming sheet, the region side where the detection portion was formed was set as the downstream side in the specimen flow direction. Next, the absorbent layer forming sheet was pasted from the downstream end of the spread layer forming sheet onto the downstream end of the support forming sheet. At this time, the absorbent layer forming sheet was disposed so as to overlap the developing layer forming sheet by 7 mm. Then, the reagent layer forming sheet was pasted on the upstream support forming sheet from the upstream end of the spreading layer forming sheet. At this time, the reagent layer forming sheet was disposed so as to overlap the developing layer forming sheet by 1 mm. Next, the sample supply layer forming sheet was laminated on the reagent layer forming sheet and an end portion of the upstream support forming sheet. The laminated sheet was cut into a strip shape having a width of 4 mm along the flow direction of the sample to prepare a sample analysis tool 107 having a width of 4 mm and a length of 77 mm. In the sample analysis tool 107, the cutting portion of the developing layer forming sheet is the developing layer 12, the cutting portion of the reagent layer forming sheet is the reagent layer 54, and the cutting portion of the sample supply layer forming sheet Is the specimen supply layer 53, and the cut portion of the absorbent layer forming sheet is the absorbent layer 56.

  The sample analysis tool 107 had an overall length of 77 mm and an overall width of 4 mm. The support 11 has a length of 77 mm and a width of 4 mm, the development layer 12 has a length of 30 mm and a width of 4 mm, the sample supply layer 53 has a length of 26 mm and a width of 4 mm, and the reagent layer 54 has a length of 10 mm and a width of 4 mm. Layer 56 was 29 mm long and 4 mm wide. In the development layer 12, the length from the upstream end to the upstream end of the detection unit 15 was 11 mm.

  Measurement was performed visually (n = 2) in the same manner as in Example 1 except that the sample analysis tool 107 was used and the sample was supplied to the sample supply layer 53. Further, as a control, the extract was dropped on the specimen supply layer 53, and similarly visually observed.

Table 1 below shows the degree of coloration determined by the following evaluation criteria for visual observation of Example 1 and Comparative Example 1. The greater the number of +, the stronger the coloring. In this example, the strongest coloring among the obtained results was evaluated as ++, and ++, +, and − were determined as relative evaluation with respect to ++.
(Evaluation criteria)
-: Colored or uncolored to the same extent as the control +: Colored ++: Stronger color +++: Strongest color

(Table 1)
Degree of visual coloration Example 1 ++
Comparative Example 1 +

  As shown in Table 1, according to the sample analysis tool of Example 1, stronger coloring was observed than the sample analysis tool of Comparative Example 1. From this result, it was found that excellent sensitivity could be achieved because the sample solution could be efficiently developed on the detection unit.

[Example 2]
In place of the blue latex-labeled FluA antibody solution, the following red latex-labeled FluB antibody solution was used, and the sample analysis tool was prepared and influenza was obtained in the same manner as in Example 1 except that type B virus was used as the sample. Virus detection was performed. As a result, the same result as in Example 1 was obtained.

  (1) of Example 1 except that red latex particles (particle size 0.3 μm, manufactured by Ceradyne) were used as latex particles, and anti-FluB monoclonal antibody (manufactured by Fitzgerald) was used as the antibody. Similarly, an antibody solution was prepared. This was designated as a red latex labeled anti-FluB antibody solution.

  According to the sample analysis tool of the present invention, it is possible to efficiently move the supplied sample solution to the detection unit by providing the sample supply layer that satisfies the above conditions. Since the sample solution can be efficiently moved in this way, for example, the amount of the sample solution can be reduced and the amount of the specimen used for preparing the sample solution can be reduced. Therefore, according to the sample analysis tool of the present invention, even a small amount of a sample derived from a living body can be efficiently moved to the detection unit with a small amount of the sample liquid, and detection with excellent sensitivity can be realized. It is. The present invention can be applied to fields such as clinical tests, biochemical tests, and medical research, for example, and its application is not limited and can be applied to a wide range of fields.

101-107 Specimen analysis tool 11 Support 12 Expanding layer 13, 53 Specimen supply layer 14, 24, 54 Reagent layer 15 Detector 16, 56 Absorbing layer

Claims (11)

A sample supply layer for supplying a sample, a reagent layer containing a labeled binding substance, a development layer for developing the specimen, a detection unit on which an immobilized binding substance is immobilized, and a support,
The spread layer, the reagent layer, and the specimen supply layer are laminated on the support,
In the flow direction of the specimen in the spreading layer,
The reagent layer is disposed in contact with the spreading layer,
The sample supply layer is disposed in a state where the downstream side surface of the sample supply layer is in contact with the upstream side surface of the reagent layer,
The spread layer includes the detection unit,
The sample analysis tool, wherein the amount of water absorption per unit volume of the sample supply layer is 5.4 mL / cm ³ or less.   The sample analysis tool according to claim 1, wherein the sample supply layer is a glass fiber member. In the flow direction of the specimen,
The upstream surface of the region of the support where the sample supply layer is laminated is exposed, or the upstream end of the support is at the same position as the upstream end of the sample supply layer Alternatively, the sample analysis tool according to claim 1, wherein the sample supply layer is disposed on the support in a state where the sample supply layer is located downstream of the upstream end of the sample supply layer. Furthermore, an absorption layer for absorbing the specimen that has expanded the expansion layer,
The absorbent layer is laminated on the support,
In the flow direction of the specimen,
The sample analysis tool according to any one of claims 1 to 3, wherein the absorption layer is disposed in a state where the absorption layer is in contact with a downstream end portion of the development layer. The sample analysis tool according to any one of claims 1 to 4, wherein a surface of the support is liquid-impermeable. The sample analysis tool according to claim 5, wherein the surface of the support is hydrophobic. The target in the sample is an antigen;
The immobilized binding substance is an immobilized antibody against the antigen;
The sample analysis tool according to any one of claims 1 to 6, wherein the labeled binding substance is a labeled antibody against the antigen. The target in the sample is an antibody;
The immobilized binding substance is an immobilized antigen for the antibody;
The sample analysis tool according to any one of claims 1 to 7, wherein the labeled binding substance is a labeled antibody against the antibody. The sample analysis tool according to any one of claims 1 to 8, wherein the label of the labeled binding substance is colored insoluble carrier particles or an enzyme. The sample analysis tool according to claim 9, wherein the colored insoluble carrier particles are colored latex particles or metal colloid particles. Using the sample analysis tool according to any one of claims 1 to 10,
By supplying a sample liquid containing a specimen to the specimen supply layer of the specimen analysis tool, and spreading the sample liquid on the development layer,
Forming a complex of the target in the sample solution and the labeled binding substance of the reagent layer;
Capturing the complex with the immobilized binding substance in the detection unit;
And a step of detecting the label of the labeled binding substance in the captured complex.

Images