Immunochromatography test strip

Abstract

The purpose of the present invention is to provide an immunochromatography test strip that can suppress the effects of hemolysis on an immunochromatography test that uses a blood sample as a specimen. An immunochromatography test strip according to the present invention includes: a chromatography medium part that is carried on an impermeable base material; and a specimen processing part that is carried on the impermeable base material at a position that is upstream of the chromatography medium part in a sample development direction. The chromatography medium part has a determination part at which a binding substance that can bind to a component to be detected is fixed, and a sample is added at the specimen processing part, which contains lectin, casein, or a salt thereof.

Description

Test strips for immunochromatography

[0001] This invention relates to a test strip for immunochromatography.

[0002] In recent years, immunoassays using immunochromatography, which do not require sample pretreatment, have become increasingly important as convenient in vitro diagnostic kits and portable diagnostic devices that utilize the specific reactivity of antibodies to detect antigens in sample solutions.

[0003] When detecting a component to be detected, consisting of an antigen in a sample, using the sandwich method by immunochromatography, the following steps are performed: (1) An antibody that specifically binds to the antigen, which is the component to be detected, is used as the immobilization reagent, and a reaction site is formed by coating a predetermined area of ​​the chromatographic medium in a predetermined manner with this antibody. (2) On the other hand, an antibody that specifically binds to the component to be detected is used as the detection reagent, and a labeled detection reagent is prepared by labeling the detection reagent with a labeling substance such as an enzyme, or by sensitizing the detection reagent with a labeling substance such as an insoluble carrier. (3) The developing solvent that constitutes the mobile phase is spread onto the chromatographic medium, which is the stationary phase, together with the sample containing the component to be detected and the labeled detection reagent.

[0004] Through the above procedure, at the reaction site formed on the chromatographic medium, the antigen, which is the component to be detected, is captured by binding to the antibody, which is the immobilized reagent fixed to the reaction site. Simultaneously, an antigen-antibody reaction occurs between the antigen and the antibody, which is the labeled detection reagent. As a result, a sandwich-type conjugate of the immobilized reagent (immobilized antibody), the component to be detected (antigen), and the detection reagent (labeled antibody) is formed at the reaction site. If the component to be detected is present in the sample, the labeling substance indirectly binds to the reaction site, causing a predetermined signal to appear, thereby enabling the detection of the component to be detected.

[0005] Immunochromatographic methods are widely used in clinical testing and laboratory measurements due to their ease of operation and rapid measurement time. While enzymes or insoluble carriers are generally used as labeling substances in immunochromatography, employing insoluble carriers (such as colloidal metal particles or colored latex particles) that can be visually detected without requiring special procedures further enhances the value of immunochromatography as a simple detection method. In recent years, methods using the aggregation of gold colloid particles as an indicator have become increasingly common.

[0006] However, not all samples can be handled identically. For example, blood contains cells such as red blood cells, white blood cells, and platelets, and these must be removed or allowed to spread to the plasma component with a delay. In particular, if red blood cells rupture, the chromatographic medium will be stained red, making it difficult to visually identify trace substances. For this reason, when using blood (whole blood) as a measurement sample, it is necessary to remove the blood cells beforehand.

[0007] Recently, methods have been developed in which plasma and serum separation pads are installed on the immunochromatographic test strip itself. For example, a blood cell separation unit is installed directly below the sample addition unit in the sample processing unit of the immunochromatographic test strip. The blood cell components are held in the blood cell separation unit, preventing them from flowing out to the chromatographic medium, and allowing the plasma components to be developed first.

[0008] In the aforementioned method, it is important to prevent the rupture of red blood cells in whole blood and to avoid hemolysis, and many methods have been proposed. Examples include a method using a carboxymethylcellulose membrane in the blood cell separation section (Patent Document 1) and a method using propanol or acrylamide (Patent Document 2).

[0009] Furthermore, a method has been proposed (Patent Document 3) for separating red blood cells using a hydrophilic sintered porous material and using them in various operations. In addition, a strip structure equipped with a membrane made of glass fibers for the purpose of separating blood cells has been proposed for the detection of HIV in the blood (Patent Document 4).

[0010] Alternatively, methods using MAP solution, or methods in which blood cells (especially red blood cells) are hemolyzed and then applied to an immunochromatographic test strip to remove the spread dye (Patent Document 5), have been proposed.

[0011] Japanese Patent Publication No. 2002-214236, Japanese Patent Publication No. 2002-350428, Japanese Patent No. 2940990, Japanese Patent Publication No. 11-248708, International Publication No. 2004 / 106930

[0012] The methods described in Patent Documents 1 to 4 and the method using the above-mentioned MAP solution cannot completely prevent hemolysis and cannot prevent the chromatographic medium from being stained red by hemoglobin. For example, if the length of the blood cell separation section is insufficient, or if the amount of antigen decreases due to blood dilution, sufficient detection sensitivity may not be ensured.

[0013] Furthermore, the method described in Patent Document 5 involves actively destroying red blood cells, which requires additional steps such as decolorization of the red-colored developed area. With such a method, even if whole blood is directly added to an immunochromatographic test strip, it is insufficient to accurately detect the target substance.

[0014] Therefore, this disclosure aims to provide an immunochromatographic test strip that can suppress the effects of hemolysis in immunochromatographic tests using blood samples as specimens.

[0015] This disclosure can be realized in the following forms: 1. An immunochromatographic test strip comprising: a chromatographic medium section having a determination section supported on an impermeable substrate and having a binding substance immobilized thereon that can bind to a component to be detected; and a sample processing section to which a sample is added, supported on the impermeable substrate at a position upstream of the chromatographic medium section in the sample development direction, wherein the sample processing section contains a lectin and casein or a salt thereof. 2. The immunochromatographic test strip according to 1, wherein the sample processing section comprises a sample pad to which a blood sample is added, a blood cell separation section to separate blood cells, and a conjugate pad containing a labeling substance, wherein the sample pad contains a lectin and casein or a salt thereof. 3. The immunochromatographic test strip according to 1 or 2, wherein the lectin is a lectin having an agglutination activity of 20 μg protein / mL or less against 2 volume percent rabbit erythrocytes. 4. The lectin content in the sample processing unit is 0.1 μg / cm³ per unit area of ​​the sample processing unit. ２ ~60 μg / cm³ ２ The immunochromatographic test strip as described in 1 or 2 above. 5. The casein or salt thereof content in the sample processing area is 20 μg / cm² per unit area of ​​the sample processing area. ２ ~2,000μg / cm ２ The immunochromatographic test strip according to 1 or 2 above. 6. The immunochromatographic test strip according to 2 above, wherein the blood sample is a blood sample containing red blood cells. 7. The immunochromatographic test strip according to 1 or 2 above, wherein the sample processing unit contains an antibody-conjugated gold nanoparticle labeling reagent. 8. The immunochromatographic test strip according to 1 or 2 above, wherein the determination unit has an antibody that recognizes the component to be detected bound to it.

[0016] The immunochromatographic test strip of this disclosure includes a chromatographic medium portion having a determination section on which a binding substance capable of binding to the component to be detected is immobilized and supported on an impermeable substrate, and a sample processing portion to which the sample is added, supported on the impermeable substrate at a position upstream of the chromatographic medium portion in the sample development direction, wherein the sample processing portion contains lectin and casein or a salt thereof. As a result, the immunochromatographic test strip of this disclosure suppresses the effect of hemolysis and enables accurate immunochromatographic testing.

[0017] Figure 1 shows the structure of an immunochromatographic test strip according to one embodiment of the present invention. Figure 2 shows one aspect of the structure of the sample processing section of the immunochromatographic test strip according to one embodiment of the present invention. Figure 3 shows the results of an immunochromatographic measurement.

[0018] The present disclosure will be described below based on embodiments, but the present disclosure is not limited to these embodiments.

[0019] The immunochromatographic test strip of this embodiment (hereinafter also referred to as "this immunochromatographic test strip") includes a chromatographic medium portion having a determination portion on which a binding substance capable of binding to the component to be detected is immobilized, and a sample processing portion to which the sample is added, which is supported on the impermeable substrate at a position upstream of the chromatographic medium portion in the sample development direction, and is characterized in that the sample processing portion contains lectin and casein or a salt thereof.

[0020] <Structure of the Sample Processing Section> In this immunochromatographic test strip, the side into which the sample is added is called the upstream or upstream side, and the direction in which the chromatograph unfolds toward the absorption pad is called the downstream or downstream side. One embodiment of this immunochromatographic test strip has the structure shown in Figure 1. One embodiment of the sample processing section has the structure shown in Figure 2. In one embodiment, it is preferable that the sample processing section contains an antibody-bound labeling reagent (antibody-bound labeling reagent), and it is more preferable that it contains an antibody-bound gold nanoparticle labeling reagent.

[0021] This immunochromatographic test strip consists of a sample processing section (6) for adding the sample and a chromatographic medium section (5), which are laminated on an impermeable substrate (4).

[0022] The sample processing section (6) can have any number of layers, as long as it has the role of reacting with the detection reagent on which the component to be detected is labeled when a blood sample is added, and capturing blood cells to suppress hemolysis into the chromatograph medium section (5). For example, 1 to 4 layers are preferred, and 2 to 3 layers are more preferred.

[0023] In one embodiment, the sample processing unit (6) is preferably composed of three layers having different properties. These three layers are a sample pad (1) to which the sample is added, a blood cell separation unit (3) to bind and separate blood cells to form plasma, and a conjugate pad (2) containing a labeling substance on which an antibody that binds to the component to be detected in the plasma is immobilized. The ends of the conjugate pad (2) and the blood cell separation unit (3) may be aligned or not.

[0024] In one embodiment, it is preferable that the conjugate pad (2) is partially laminated on the chromatograph medium section (5), and that the blood cell separation section (3) is further laminated via the conjugate pad (2). The arrangement of the conjugate pad (2) and the blood cell separation section (3) is not particularly limited, and either may be placed on top of the other. Furthermore, the sample pad (1) is laminated on the uppermost side via the conjugate pad (2). This section with a three-layer structure is called the specimen processing section (6).

[0025] In the above embodiment, in the sample processing unit (6), a blood sample is added to the sample pad (1), the blood cell separation unit (3) separates the plasma from the blood cells, the components to be detected are specifically bound to a label by an antigen-antibody reaction in the conjugate pad (2) to form a complex, the complex moves together with the plasma from the conjugate pad (2) to the chromatograph medium unit (5) by capillary action, spreads out on the chromatograph medium unit (5), stops in the determination unit on the chromatograph medium unit (5) and is detected. The determination unit is not shown in Figure 1, but can be formed at any position on the chromatograph medium unit (5).

[0026] In the present disclosure, a blood sample means whole blood, plasma, serum, a dilution of blood with a developing solution, or a processed sample containing these. Note that the suppression of the influence of hemolysis, which is an effect of the present invention, is particularly remarkable in a sample containing red blood cells (for example, whole blood or diluted whole blood).

[0027] <<Sample Pad (1)>> The sample pad (1) temporarily absorbs and holds the sample. Examples of the material constituting the sample pad (1) include membranes made of glass fiber, synthetic fibers such as polyacrylic fiber and polyethylene fiber, dry paper, paper pulp, woven fabric, regenerated cellulose fibers such as rayon and cupra, or a combination thereof.

[0028] (Lectin) In the test strip for this immunochromatograph, the specimen treatment part (6) contains lectin and casein or a salt thereof. The content of lectin in the specimen treatment part (6) is preferably 0.1 μg / cm ２ to 60 μg / cm ２ per unit area of the specimen treatment part.

[0029] The content of lectin in the specimen treatment part (6) is preferably 0.1 μg / cm ２ or more per unit area of the specimen treatment part, more preferably 0.4 μg / cm ２ or more, and even more preferably 0.9 μg / cm ２ or more. When the content of the lectin is 0.1 μg / cm ２ or more, the effect of suppressing the influence of hemolysis can be enhanced. Lectin has the effect of aggregating red blood cells to form aggregates, and these aggregates stay in each pad or membrane without passing through the porous structure of the specimen treatment part (6) [such as the sample pad (1), conjugate pad (2), blood cell separation part (3), etc.]. Therefore, blood cells will not be developed near the reaction site of at least the chromatographic medium part (5) on the downstream side of the test strip for immunochromatography, and thus hemolysis can be effectively suppressed in an immunochromatographic test using a blood sample as the specimen.

[0030] The lectin content in the specimen processing unit (6) is preferably 60 μg / cm per unit area of the specimen processing unit ２ or less, more preferably 30 μg / cm ２ or less, and even more preferably 15 μg / cm ２ or less. By having the lectin content of 60 μg / cm ２ or less, an effect of suppressing hemolysis can be expected without causing an unexpected non-specific reaction due to excessive lectin within an economically feasible range.

[0031] In the test strip for this immunochromatograph, the lectin preferably has an agglutination activity of 40 μg protein / mL or less against 2 volume percent of rabbit red blood cells. The agglutination activity is more preferably 20 μg protein / mL or less, and even more preferably 10 μg protein / mL or less. By having the agglutination activity of 40 μg protein / mL or less, the effect of suppressing the influence of hemolysis can be enhanced. The lower limit value of the agglutination activity is not particularly limited, but is, for example, 2 μg protein / mL or more.

[0032] A specific method for measuring the agglutination activity of lectins includes the following steps (x1) to (x6): (x1) Take 2-3 mL of stored blood into a screw-cap test tube, add 4-5 times the volume of PBS, centrifuge at 2,000 rpm for 5 minutes, and discard the supernatant. Repeat this operation 3-4 times to obtain a red blood cell fraction from the stored blood. (x2) Add PBS to the red blood cell fraction obtained in (x1) to make a 2-volume percent red blood cell suspension. (x3) Dispense 25 μL of PBS into the 2nd to 11th rows of each well in a 96-well titer plate (U-bottom). (x4) Place 50 μL of lectin solution into the 1st row of the 96-well titer plate, take 25 μL of the lectin solution from the 1st row, transfer it to the well to the right (2nd row), and mix by pipetting. Repeat this operation up to the 11th row to create a 2-fold dilution row. (x5) Dispense 50 μL of 2% (v / v) red blood cell suspension into rows 1-11 and leave at room temperature for 30-60 minutes. (x6) Determine the presence or absence of agglutination by observing the sedimentation of red blood cells at the bottom of each well. Activity is indicated by the final concentration on the titer plate, which is the lowest concentration of lectin that can agglutinate red blood cells.

[0033] Examples of lectins in this embodiment include plant-derived lectins. Examples of plant-derived lectins include Lens culinaris aglutinin (LCA: derived from lentils), Phaseolus Vulgaris Erythroaglutinin (PHA-E: derived from kidney beans), Ricinus communis aglutinin (RCA: derived from castor beans), Wheat germ aglutinin (WGA: derived from wheat germ), Agaricus bisporusaaglutinin (ABA: derived from mushrooms), and Sambucus sieboldiana aglutinin (SSA: derived from elderberry). Among these, PHA-E, ABA, and SSA are preferred because they are known to recognize complex branched sugar chains and their modification forms (sialic acid / fucose modification) (ACS Chemical Biology Vol 17, Issue 11 2022).

[0034] As the structure of the lectin, it preferably has a carboxyl terminus, a calcium-binding site, and a sugar-binding domain, and has a dimer structure. By having a carboxyl terminus (C-terminus), the binding ability to sugar chains can be enhanced. By having a calcium-binding site, the stability of the lectin structure and the binding to sugar chains can be improved. By having a sugar-binding domain, it shows affinity for a specific sugar chain pattern and can enhance the blood agglutination activity. Further, by having a dimer structure, the binding to sugar chains and the agglutination action can be improved.

[0035] The molecular weight of the lectin varies depending on the type and origin of the lectin, but it is preferably 10,000 to 200,000 daltons (Da), more preferably 12,000 to 180,000 Da, and even more preferably 16,000 to 160,000 Da. When the molecular weight of the lectin is within the above range, sufficient blood agglutination activity can be exhibited.

[0036] (Casein) In the test strip for this immunochromatograph, the content of casein or its salt in the sample treatment part (6) is preferably 20 μg / cm ２ to 2,000 μg / cm ２ per unit area of the sample treatment part.

[0037] The content of casein or its salt in the sample treatment part (6) is preferably 20 μg / cm ２ or more per unit area of the sample treatment part, more preferably 50 μg / cm ２ or more, and even more preferably 100 μg / cm ２ or more. When the content of the casein or its salt is 20 μg / cm ２ or more, the effect of suppressing the influence of hemolysis can be enhanced.

[0038] The content of casein or its salt in the sample treatment part (6) is preferably 2,000 μg / cm ２ or less per unit area of the sample treatment part, more preferably 1,000 μg / cm ２ or less, and even more preferably 500 μg / cm ２ or less. When the content of the casein or its salt is 2,000 μg / cm ２The following method makes the process easier and more economical in terms of casein solubility. Furthermore, it can suppress problems such as delayed development when blood samples are spread onto immunochromatographic test strips due to excessive casein.

[0039] Casein is a protein extracted from dairy products, primarily existing in milk as spherical structures called micelles. Casein salts (caseinates) are obtained by treating casein with alkali metals (sodium, potassium) or ammonium.

[0040] The effects of including casein together with lectin in the sample processing unit (6) will be explained below. When a blood sample is added to the sample processing unit (6) containing lectin, red blood cells that are deployed near the lectin contained in the sample processing unit (6) come into contact with the lectin and begin to form aggregates. On the other hand, there are red blood cells and red blood cell fragments that are not present near the lectin and are hemolyzed by coming into contact with the components of the sample processing unit (6) before coming into contact with the lectin.

[0041] By introducing casein together with lectin in the sample processing unit (6), hemolyzed red blood cells and red blood cell fragments are captured by the casein to form agglutination clumps, and the agglutination reaction between lectin and red blood cells is promoted by the casein. This is thought to prevent red blood cells that were unable to form agglutination clumps with lectin, as well as hemolyzed red blood cells and red blood cell fragments, from flowing out of the sample processing unit (6) and spreading into the chromatograph medium unit (5). Therefore, the effect of hemolysis in the chromatograph medium unit (5) can be suppressed more effectively, especially when using blood samples containing red blood cells. As a result, high discriminability can be maintained even during relatively long detection times of about 30 minutes.

[0042] Furthermore, casein, together with lectin, forms agglutinations of red blood cells and contributes to the formation of complexes containing casein, lectin, red blood cells, and hemolyzed red blood cells or red blood cell fragments that are larger than the pore size of the blood cell separation section (3). Due to this action, by including casein together with lectin in the sample processing section (6), the effect of hemolysis in the chromatographic medium section (5) can be suppressed even when a blood sample is added, and the discriminability in the detection section can be improved even in tests where the result time is long.

[0043] The sample processing unit (6) [in one embodiment, in particular, the sample pad (1)] may contain a spreading aid. Examples of spreading aids include blocking agents, surfactants, buffers, and protease inhibitors. Another example is mannitol. Hemolysis can be suppressed by including mannitol.

[0044] Examples of blocking agents include bovine serum albumin (BSA), skim milk powder, gelatin, synthetic polymers, and heat-denatured proteins.

[0045] Examples of surfactants include polyoxyethylene sorbitan monooleate 80, polyoxyethylene sorbitan monooleate 20, and octylphenol ethylene oxide adducts.

[0046] Examples of buffers include phosphate buffer solutions (e.g., PBS) and Tris buffer solutions (e.g., TBS). Examples of protease inhibitors include phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA).

[0047] <<Conjugate Pad (2)>> Examples of materials constituting the conjugate pad (2) include glass fiber nonwoven fabrics, cellulose nonwoven fabrics, polyethylene, polypropylene, polyester, and other synthetic polymer fiber nonwoven fabrics. The material is not particularly limited as long as it can hold and dry the labeled detection reagent and does not inhibit the redissolution of the labeled detection reagent into the developing solution when a sample containing the component to be detected is developed during testing.

[0048] <<Blood Cell Separation Unit (3)>> The blood cell separation unit (3) only needs to have the function of separating blood cell components such as red blood cells, white blood cells, and platelets, or drastically reducing their flow rate, thereby preferentially spreading plasma components onto the membrane. Examples of materials constituting the blood cell separation unit (3) include synthetic polymer fibers such as cellulose and polyamide, and membranes such as thin filter paper made of glass fiber fibers.

[0049] The membrane only needs to be capable of separating red blood cells contained in the whole blood or diluted whole blood sample, and preferably has the function of preferentially spreading plasma components onto the membrane by separating blood cell components such as white blood cells and platelets or by drastically reducing their spread rate.

[0050] Examples of the aforementioned membrane include synthetic polymer fibers functionalized with hydroxyl groups, amino groups, carboxymethyl groups, etc., and glass fibers to which synthetic polymer alcohols such as polyvinyl alcohol are bonded.

[0051] <<Chromatography Medium Section (5)>> The chromatography medium section (5) used in this immunochromatography test strip has a determination section supported on an impermeable substrate, on which a binding substance that specifically binds to the component to be detected is immobilized. In one embodiment, it is preferable that an antibody that recognizes the component to be detected (a component-recognizing antibody) is bound to the determination section.

[0052] In this embodiment, the chromatographic medium (5) is an inert material made of a finely porous substance that exhibits capillary action and does not react with the detection reagent, immobilization reagent, or component to be detected used. The material is not particularly limited as long as it has a development speed that allows for sufficient sensitivity to be obtained in a short time.

[0053] In this embodiment, the chromatograph medium (5) can be, for example, a fibrous or nonwoven fibrous matrix composed of ceramic fine particles such as silica, titania, zirconia, ceria, and alumina, or fine particles of organic polymers, or a cellulose derivative such as polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, nitrocellulose, or cellulose acetate, or a film, filter paper, glass fiber filter paper, cloth, cotton, etc.

[0054] Even if the fine particles themselves are not porous, when packed, voids are created between the fine particles and they function as a chromatographic medium (5). Preferably, these are cellulose derivatives, nylon films, filter paper, glass fiber filter paper, etc., and more preferably nitrocellulose films, mixed nitrocellulose ester (a mixture of nitrocellulose and cellulose acetate) films, nylon films, or filter paper.

[0055] The shape and size of the chromatographic medium (5) used in this immunochromatographic test strip are not particularly limited, and should be appropriate in terms of actual operation and observation of reaction results. To simplify the operation, it is preferable to provide a support such as an impermeable substrate made of plastic on the back surface of the chromatographic medium (5) on which the reaction site is formed. The properties of this impermeable substrate are not particularly limited, but when observing the measurement results by visual inspection, it is preferable that the impermeable substrate has a color that is distinguishable from the color provided by the labeling substance, and is usually colorless or white.

[0056] <Detection Unit> On the chromatographic medium (5) used in this immunochromatographic test strip, a detection unit (reaction site) is formed in which a substance that specifically binds to the component to be detected, such as an antibody, is immobilized as an immobilization reagent at an arbitrary position. Although the detection unit is not shown in Figure 1, it can be formed at any position on the chromatographic medium (5). Methods for immobilizing the immobilization reagent on the chromatographic medium (5) include a method of directly immobilizing the immobilization reagent on the chromatographic medium (5) by physical or chemical means, and an indirect immobilization method in which the immobilization reagent is physically or chemically bound to insoluble microparticles such as latex particles, and these microparticles are captured and immobilized on the chromatographic medium (5).

[0057] Methods for direct immobilization include physical adsorption and covalent bonding. Generally, physical adsorption can be performed when the chromatographic medium (5) is a nitrocellulose membrane or a mixed nitrocellulose ester membrane.

[0058] In covalent bonding, cyanide bromide, glutaraldehyde, carbodiimide, etc., are generally used to activate the chromatographic medium (5), but any of these methods may be used. As an indirect immobilization method, an immobilization reagent is bound to insoluble microparticles, and then immobilized on the chromatographic medium (5).

[0059] The particle size of the insoluble fine particles can be selected to be such that they are captured by the chromatographic medium (5) but cannot move, and preferably they are fine particles with an average particle size of 10 μm or less. Various types of particles used in antigen-antibody reactions are known and can be used in this embodiment as well.

[0060] Examples of insoluble microparticles include microparticles of organic polymer substances such as organic polymer latex particles obtained by emulsion polymerization methods, such as polystyrene, styrene-butadiene copolymer, styrene-methacrylate copolymer, polyglycidyl methacrylate, and acrolein-ethylene glycol dimethacrylate copolymer; microparticles of gelatin, bentonite, agarose, and crosslinked dextran; inorganic oxides such as silica, silica-alumina, and alumina, and inorganic particles obtained by introducing functional groups into inorganic oxides through silane coupling treatment, etc.

[0061] In this embodiment, direct immobilization is preferred due to the ease of sensitivity adjustment, etc. Furthermore, conventionally known methods can be used to immobilize the reagent onto the chromatographic medium (5). For example, various techniques such as microsyringes, pens with adjustable pumps, and ink spray printing can be used. The morphology of the reaction site is not particularly limited, but it can be immobilized as a circular spot, a line perpendicular to the development direction of the chromatographic medium (5), numbers, letters, or symbols such as + and -.

[0062] After immobilizing the immobilization reagent, the chromatograph medium (5) may be blocked by known methods as needed to prevent a decrease in analytical accuracy due to nonspecific adsorption. Generally, proteins such as bovine serum albumin, skim milk, casein, and gelatin are preferably used for blocking. After such blocking, the medium may be washed with one or more surfactants such as polyoxyethylene sorbitan monooleate 20, octylphenol ethylene oxide additive, and sodium dodecyl sulfate (SDS) as needed.

[0063] In addition, the chromatograph medium (5) may include, as needed, a sample addition area (sample pad, etc.) for adding a sample containing the component to be detected, an area for removing solid components such as blood cells from the sample (blood cell separation area, etc.), a developing solution addition area for adding developing solution, an absorption area (absorption pad, etc.) for absorbing the component to be detected and developing solution that were not captured by the reaction area, and a control area to indicate that the measurement was performed correctly. The materials of these areas are not particularly limited as long as the sample solution and developing solution can be moved by capillary action. Generally, a suitable material can be selected from a plurality of porous materials such as nitrocellulose membranes, filter paper, and glass fiber filter paper, and they can be arranged to be connected by capillary action to the chromatograph medium (5) on which the immobilized reagent is immobilized.

[0064] <Labeling Substance> The detection reagent used in this embodiment is a substance that specifically binds to the component to be detected, such as an antibody, and is labeled with a labeling substance. Enzymes are generally used to label detection reagents in immunochromatography, but an insoluble carrier is used as the labeling substance in this embodiment because it is suitable for visually determining the presence of the component to be detected. In this embodiment, a labeled detection reagent is prepared by sensitizing the detection reagent to an insoluble carrier.

[0065] Examples of insoluble carriers used as labeling substances in this embodiment include colloidal metal particles such as gold, silver, and platinum; colloidal metal oxide particles such as iron oxide; colloidal nonmetal particles such as sulfur; latex particles made of synthetic polymers; and others. Colloidal gold particles are particularly preferred because they are easy to detect and have excellent visibility.

[0066] Insoluble carriers are labeling materials suitable for visually determining the presence of the component to be detected, and are preferably colored to facilitate visual determination. Colloidal metal particles and colloidal metal oxide particles themselves exhibit a specific natural color corresponding to their particle size, and their color can be used as a label.

[0067] Since latex particles made of synthetic polymers are naturally white, they cannot be used as labeling materials as they are. However, by dyeing them with oil-soluble dyes, particularly latex particles in an aqueous medium, using an emulsion of oil-soluble dyes dissolved in an oily organic solvent, they can be made to have a desired color and intensity.

[0068] The latex particles that can be used as labeling substances in this embodiment can be prepared by polymerizing or copolymerizing various monomers. Examples of monomers include polymerizable unsaturated aromatics such as styrene, chlorostyrene, α-methylstyrene, divinylbenzene, and vinyltoluene; polymerizable unsaturated carboxylic acids such as (meth)acrylic acid, itaconic acid, maleic acid, and fumaric acid; polymerizable unsaturated carboxylic acid esters such as methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, glycidyl (meth)acrylate, ethylene glycol-di-(meth)acrylate ester, and tribromophenyl (meth)acrylate; unsaturated carboxylic acid amides such as (meth)acrylonitrile, (meth)acrolein, (meth)acrylamide, N-methylol-(meth)acrylamide, methylenebis(meth)acrylamide, butadiene, isoprene, vinyl acetate, vinylpyridine, N-vinylpyrrolidone, vinyl chloride, vinylidene chloride, and vinyl bromide; polymerizable unsaturated nitriles; vinyl halides; and conjugated dienes. These monomers are appropriately selected based on the surface properties, specific gravity, etc., required for use as a labeling substance, and can be used individually or in combination of two or more.

[0069] In this embodiment, particularly preferred latex particles as labeling substances include, for example, copolymers of styrene and methacrylic acid, and copolymers of styrene and itaconic acid. Examples of polymerization initiators for polymerization reactions to obtain such copolymers include, for example, persulfates. The average particle size of the latex particles used as labeling substances is preferably in the range of 50 to 500 nm.

[0070] Colloidal metal particles and colloidal metal oxide particles that can be used as labeling substances in this embodiment include, for example, colloidal gold particles, colloidal silver particles, colloidal platinum particles, colloidal iron oxide particles, and colloidal aluminum hydroxide particles. In particular, colloidal gold particles and colloidal silver particles are preferred in that, at appropriate particle sizes, colloidal gold particles exhibit a red color and colloidal silver particles exhibit a yellow color. The average particle size of these colloidal metal particles is preferably in the range of 1 to 500 nm, more preferably 10 nm to 150 nm, and more preferably 20 to 100 nm, in order to obtain a particularly strong color tone.

[0071] It is known that the surfaces of these insoluble carriers, whether latex particles or colloidal metal particles, are negatively charged (see, for example, Japanese Patent Publication No. 5-133956). For example, in colloidal metal particles, anions derived from the reducing agent added during the manufacturing process are adsorbed on their surface, preventing mutual aggregation and maintaining a dispersed state. It is known that when a low concentration of surfactant that does not neutralize the surface charge is added to colloidal metal particles in this state, the particles aggregate into a chain of several units (Japanese Patent Publication No. 2006-58781).

[0072] Thus, in the immunochromatographic method of this embodiment, when a vinyl-based water-soluble polymer having an oxygen atom-containing polar group and a nonionic surfactant are added to the developing solvent constituting the mobile phase, it is presumed that a few insoluble carriers trapped at the reaction site on the chromatographic medium aggregate, thereby amplifying the positive signal observed at the reaction site.

[0073] In particular, with colloidal metal particles, it is presumed that the increase in the number of particles accumulated at the reaction site due to aggregation not only increases the amount of signal that can be visually determined, but also changes in the optical absorption spectral characteristics of the particles, making it possible to obtain a clearer positive signal at the reaction site. Because of these advantages, colloidal precious metal particles, especially colloidal gold particles, are preferred as labeling materials in this embodiment.

[0074] When using colloidal metal particles, for example, colloidal gold particles, commercially available ones may be used. Alternatively, colloidal gold particles can be prepared by conventional methods, such as reducing chloroauric acid with sodium citrate.

[0075] Known methods such as physical adsorption and chemical bonding can be used to sensitize colloidal metal particles with the detection reagent used in this embodiment. For example, a detection reagent sensitized with antibody to colloidal gold particles can be prepared by adding antibody to a solution in which gold particles are dispersed colloidally, allowing for physical adsorption, and then adding bovine serum albumin solution to block the surface of particles to which the antibody is not bound.

[0076] In actual immunochromatographic procedures, the detection reagent labeled with an insoluble carrier can be applied by dispersing it in the developing solvent that constitutes the mobile phase, or it can be applied by placing it on the mobile phase's development path in the chromatographic medium that constitutes the stationary phase, that is, in the region between the end of the chromatographic medium (5) to which the mobile phase is applied and the reaction site.

[0077] When the detection reagent is placed on the chromatographic medium, it is preferable to support the detection reagent so that it can quickly dissolve in the developing solution and move freely by capillary action. To improve the resolubility of the insoluble carrier sensitized with the detection reagent, the support area may be coated with sugars such as saccharose, maltose, lactose, or sugar alcohols such as mannitol, or pre-coated with these substances.

[0078] When the detection reagent is placed on the chromatographic medium by coating, drying, etc., it can be directly coated and dried on the chromatographic medium (5) on which the immobilized reagent is immobilized, or it can be coated and dried on another porous material, such as cellulose filter paper, glass fiber filter paper, or nylon nonwoven fabric, to form a conjugate pad, and then positioned so as to be connected to the chromatographic medium (5) on which the immobilized reagent is immobilized by capillary action.

[0079] <<Detectable Components>> The detectable components detected by the method of this embodiment are not particularly limited as long as there is a substance that specifically binds to them, and examples include proteins, peptides, nucleic acids, sugars (especially the sugar chain portion of glycoproteins, the sugar chain portion of glycolipids, etc.), and complex carbohydrates.

[0080] In this specification, "specifically binding" means binding based on the affinity between biomolecules. Examples of such affinity-based binding include binding between antigens and antibodies, binding between glycans and lectins, binding between hormones and receptors, binding between enzymes and inhibitors, and binding between complementary nucleic acids and between nucleic acids and nucleic acid-binding proteins. Therefore, when the component to be detected is antigenic, examples of substances that specifically bind to the component to be detected include polyclonal antibodies or monoclonal antibodies. Also, when the component to be detected is a glycan, examples of substances that specifically bind to the component to be detected include lectin proteins.

[0081] Specific components to be detected include, but are not limited to, carcinoembryonic antigen (CEA), HER2 protein, Pentraxin (PTX3), prostate-specific antigen (PSA), sepsis markers, CA19-9, alpha-fetoprotein (AFP), immunosuppressive acid protein (IPA), CA15-3, CA125, estrogen receptor, progesterone receptor, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), syphilis antibody, influenza virus, human hemoglobin, Chlamydia antigen, group A β-hemolytic streptococcus antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, and glycated albumin.

[0082] The immunochromatographic test strip, consisting of the above components, can be used as is, or, if necessary, placed in a molded plastic housing case.

[0083] <<Developing Solution>> The developing solution used in this embodiment is a liquid that constitutes the mobile phase in the immunochromatographic method, and moves along the chromatographic medium (5), which is the stationary phase, together with the sample containing the component to be detected and the labeled detection reagent. The developing solution is not particularly limited as long as it can develop stably without impairing the reactivity of the sample and detection reagent.

[0084] As described above, the following configurations are disclosed in this specification: 1. An immunochromatographic test strip comprising: a chromatographic medium section having a determination section supported on an impermeable substrate and having a binding substance immobilized thereon that can bind to a component to be detected; and a sample processing section to which a sample is added, supported on the impermeable substrate at a position upstream of the chromatographic medium section in the sample development direction, wherein the sample processing section contains a lectin and casein or a salt thereof. 2. The immunochromatographic test strip according to 1, wherein the sample processing section comprises a sample pad to which a blood sample is added, a blood cell separation section to separate blood cells, and a conjugate pad containing a labeling substance, wherein the sample pad contains a lectin and casein or a salt thereof. 3. The immunochromatographic test strip according to 1 or 2, wherein the lectin is a lectin having an agglutination activity of 20 μg protein / mL or less against 2 volume percent rabbit erythrocytes. 4. The lectin content in the sample processing unit is 0.1 μg / cm³ per unit area of ​​the sample processing unit. ２ ~60 μg / cm³ ２ The immunochromatographic test strip according to any one of 1 to 3 above. 5. The casein or salt thereof content in the sample processing area is 20 μg / cm² per unit area of ​​the sample processing area. ２ ~2,000μg / cm ２1. An immunochromatographic test strip according to any one of 1 to 4 above. 6. An immunochromatographic test strip according to any one of 2 to 5 above, wherein the blood sample is a blood sample containing red blood cells. 7. An immunochromatographic test strip according to any one of 1 to 6 above, wherein the sample processing unit contains an antibody-conjugated gold nanoparticle labeling reagent. 8. An immunochromatographic test strip according to any one of 1 to 7 above, wherein the determination unit has an antibody that recognizes the component to be detected bound to it.

[0085] The present invention will be described below based on examples, but the present invention is not limited thereto.

[0086] 1. Preparation of test strips for immunochromatography (1) Preparation of the chromatographic medium A sheet HF240 made of nitrocellulose (Millipore: 300 mm x 25 mm) was used as the chromatographic medium. Anti-PSA monoclonal antibody (first antibody) was diluted to a concentration of 0.75 mg / mL in carbonate buffer (pH 9.0) containing 5% by mass of isopropyl alcohol. 40 μL of this solution was spread on the membrane in a width of 1 mm and dried overnight at 60°C to create a test line on the chromatographic medium. For the control line, 40 μL of a solution prepared by diluting goat anti-mouse IgG antibody to a concentration of 1.0 mg / mL in carbonate buffer (pH 9.0) containing 5% by mass of isopropyl alcohol was spread on the membrane in a width of 1 mm downstream of the test line in the development direction.

[0087] (2) Preparation of the Labeled Substance Solution To 0.5 mL of a gold colloid dispersion (manufactured by Tanaka Kikinzoku Kogyo Co., Ltd.: LC 40 nm), 0.1 mL of anti-PSA monoclonal antibody (second antibody), diluted to a concentration of 0.025 mg / mL with Biciné buffer (pH 9.0), was added and allowed to stand at room temperature for 10 minutes. Next, 0.1 mL of MES buffer (pH 6.0) containing 0.01% by mass of methoxy-PEG-thiol 5000 (manufactured by Nippon Oil & Fats Co., Ltd., product name: SUNBRIGHT ME-050SH, molecular weight 5000) was added and allowed to stand at room temperature for another 10 minutes. After that, the mixture was thoroughly stirred, centrifuged at 8000 × g for 15 minutes, and the supernatant was removed. Then, 0.1 mL of Tris-HCl buffer (pH 8.5) containing 0.1% by mass of bovine serum albumin was added to disperse the labeled substance. The labeled substance solution was prepared using the above procedure.

[0088] (3) Preparation of conjugate pads 300 μL of the labeling reagent solution prepared above was mixed with 300 μL of 10% trehalose aqueous solution and 600 μL of distilled water. This mixture was then uniformly added to an 8 mm x 300 mm fiberglass pad (SureWick, trade name, manufactured by Millipore), and dried in a vacuum dryer to prepare the conjugate pads.

[0089] (4) Preparation of sample pads Using a 16 mm x 300 mm fiberglass pad (SureWick, trade name, manufactured by Millipore), 2.4 mL of 50 mM phosphate buffer (pH 8.0) containing 1% by mass of Tween® 80 and 5% by mass of mannitol, and 0.0075 mg / mL of SSA, ABA, PHA-E4 (manufactured by MGC) or 0.5% by mass of sodium caseinate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as shown in Table 1 was uniformly absorbed, and then the pad was freeze-dried for 4 hours to prepare the sample pads.

[0090] (5) Blood cell separation section A GF / DVA (trade name, manufactured by Whatman: 180 mm x 30 mm) was used as the blood cell separation section.

[0091] (6) Preparation of immunochromatographic test strips A substrate made of an impermeable substrate with adhesive applied to one side was to which the chromatographic medium section (5), blood cell separation section (3), conjugate pad (2), sample pad (1), and absorbent pad (7) for absorbing the unfolded sample and labeling substance prepared above were attached. Then, it was cut with a cutting machine to a width of 5 mm to make an immunochromatographic test strip.

[0092] 2. Immunochromatographic Measurement (Detection of PSA in Blood Using Human Whole Blood) PSA in blood was detected using the prepared immunochromatographic test strips. Specifically, a negative sample with a PSA concentration of 1 ng / mL and a positive sample with a PSA concentration of 30 ng / mL were used as subjects. 150 μL of each subject was placed on the sample pad of the immunochromatographic test strip and allowed to unfold, and the result was visually determined after 30 minutes. In Table 1, PSA is represented as "antigen".

[0093] PSA is detectable in very small amounts even in healthy individuals, and its normal range is generally 4 ng / mL. A PSA level of 10 ng / mL is considered to indicate a 50% or greater probability of prostate cancer. In the examples described herein, as stated above, a sample with a PSA concentration of 1 ng / mL in the blood is considered a negative sample, and a sample with a PSA concentration of 30 ng / mL is considered a positive sample.

[0094] In the presence of the antigen, negative and positive samples were tested three times each under different conditions and visually evaluated. The number of times hemolysis occurred out of the three tests was recorded as the "number of times hemolysis was observed," and an overall evaluation was made according to the following criteria. The results are shown in Table 1 and Figure 3. (For negative samples with a PSA concentration of 1 ng / mL in the blood) A1: No hemolysis was observed, and no color development was seen on the test line. B1: Some hemolysis was observed, but no color development was seen on the test line. C1: Hemolysis was present, and the presence of the test line could not be identified. (For positive samples with a PSA concentration of 30 ng / mL) A2: No hemolysis was observed, and the red line of the test line could be seen. B2: Some hemolysis was observed, but the red line of the test line could be seen. C2: Hemolysis was present, and the red line of the test line could not be seen.

[0095] The reagents shown in Table 1 are described below. SSA: Japanese elderberry lectin (Agglutination activity <20 μg protein / mL) manufactured by MGC Corporation. ABA: Mushroom lectin (Agglutination activity <20 μg protein / mL) manufactured by MGC Corporation. PHA-E4: Kidney bean lectin-E4 (Agglutination activity <10 μg protein / mL) manufactured by MGC Corporation.

[0096]

[0097] As shown in Table 1, the immunochromatographic test strip of this embodiment, in which the sample pad contains lectin and casein or a salt thereof, was confirmed to be able to detect with high sensitivity without being affected by hemolysis. In contrast, the immunochromatographic test strip that does not contain lectin and casein or a salt thereof was unable to detect the color development in the detection area due to hemolysis, or the hemoglobin partially seeped into the chromatographic medium, causing the background to be red even if it was not completely stained red, making it difficult to distinguish from the color development in the detection area, resulting in a decrease in detection sensitivity.

[0098] Furthermore, as shown in Figure 3, in immunochromatographic test strips that did not contain lectin and casein or its salts, the chromatographic medium portion was stained red with hemoglobin up to the control line, making it impossible to distinguish whether or not color development occurred due to the binding of the labeling substance. However, in the immunochromatographic test strip of this embodiment, there was no hemoglobin staining in the chromatographic medium portion, and the color development of the test line and control line due to the binding of the labeling substance could be clearly observed in a state close to that before the sample was added.

[0099] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications are possible without departing from the spirit and scope of the invention. This application is based on Japanese Patent Application No. 2024-189352, filed on 28 October 2024, which is incorporated by reference in its entirety. All references incorporated herein are incorporated as a whole.

[0100] 1. Sample pad 2. Conjugate pad 3. Blood cell separation section 4. Impermeable substrate 5. Chromatography medium section 6. Sample processing section 7. Absorbent pad

Claims

1. An immunochromatographic test strip comprising: a chromatographic medium section having a determination section supported on an impermeable substrate and having a binding substance immobilized thereon that can bind to a component to be detected; and a sample processing section to which a sample is added, supported on the impermeable substrate at a position upstream of the chromatographic medium section in the sample development direction, wherein the sample processing section contains lectin and casein or a salt thereof.

2. The immunochromatographic test strip according to claim 1, wherein the sample processing unit comprises a sample pad to which a blood sample is added, a blood cell separation unit for separating blood cells, and a conjugate pad containing a labeling substance, and the sample pad contains lectin and casein or a salt thereof.

3. The immunochromatographic test strip according to claim 1 or 2, wherein the lectin is a lectin having an agglutination activity of 20 μg protein / mL or less against 2 volume percent rabbit erythrocytes.

4. The lectin content in the sample processing unit is 0.1 μg / cm² per unit area of ​​the sample processing unit. ２ ~60 μg / cm³ ２ The immunochromatographic test strip according to claim 1 or 2.

5. The casein or salt content in the sample processing unit is 20 μg / cm² per unit area of ​​the sample processing unit. ２ ~2,000μg / cm ２ The immunochromatographic test strip according to claim 1 or 2.

6. The immunochromatographic test strip according to claim 2, wherein the blood sample is a blood sample containing red blood cells.

7. The immunochromatographic test strip according to claim 1 or 2, wherein the sample processing unit contains an antibody-conjugated gold nanoparticle labeling reagent.

8. The immunochromatographic test strip according to claim 1 or 2, wherein the determination unit has an antibody that recognizes the component to be detected bound to it.

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