Test strips for immunochromatography

Abstract

The objective is to provide an immunochromatographic test strip that can suppress the effects of hemolysis in immunochromatographic tests using blood samples. [Solution] An immunochromatographic test strip comprising a chromatographic medium section having a determination section on which a binding substance capable of binding to a component to be detected is immobilized, supported on an impermeable substrate, 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.

Description

[Technical Field]

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

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

[0003] When detecting a substance to be detected consisting of an antigen in a sample using the sandwich method by immunochromatography, the following procedure is performed. (1) An antibody that specifically binds to the antigen to be detected is used as an immobilization reagent, and this immobilization reagent is applied in a predetermined manner to a predetermined area of ​​the chromatographic medium, thereby forming a reaction site at any position on the chromatographic medium. (2) Alternatively, an antibody that specifically binds to the substance 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 solution that constitutes the mobile phase is spread onto the chromatographic medium, which is the stationary phase, together with the sample containing the substance to be detected and the labeled detection reagent.

[0004] Through the above procedure, at the reaction site formed in the chromatographic medium, the antigen, which is the substance 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 substance to be detected (antigen), and the detection reagent (labeled antibody) is generated at the reaction site. When the substance 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 substance 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, and the blood cell components are held in the blood cell separation unit, preventing the blood cell components 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, a method using MAP solution, a method of subjecting a test strip for immunochromatography to hemolysis of blood cells (particularly red blood cells) and then removing the developed dye (Patent Document 5) have been proposed.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0012] The methods described in Patent Documents 1 to 4 and the method using the above MAP solution cannot necessarily prevent hemolysis and cannot prevent the chromatographic medium part from turning red due to hemoglobin. For example, in cases where a sufficiently long blood cell separation part cannot be ensured, or when the antigen amount decreases due to blood dilution and sufficient detection sensitivity cannot be ensured.

[0013] Also, in the method described in Patent Document 5, hemolysis of red blood cells is actively carried out, but for this purpose, operations and extra operations such as decolorization of the developed red-colored part are required. Thus, it is insufficient to directly add whole blood to a test strip for immunochromatography and accurately detect the target substance.

[0014] Therefore, the present disclosure aims to provide an immunochromatographic test strip capable of suppressing the influence of hemolysis in an immunochromatographic test using a blood sample as a specimen.

Means for Solving the Problems

[0015] The present disclosure can be realized in the following forms. 1. A chromatographic medium part having a determination part on an impermeable base material, where a binding substance capable of binding to a detected component is immobilized, and An immunochromatographic test strip including a specimen treatment part where a specimen to which a sample is added is supported on the impermeable base material at a position upstream in the sample development direction from the chromatographic medium part, The immunochromatographic test strip, wherein the specimen treatment part contains lectin and casein or a salt thereof. 2. The specimen treatment part is composed of a sample pad to which a blood sample is added, a blood cell separation part for separating blood cells, and a conjugate pad containing a labeling substance, The immunochromatographic test strip according to 1 above, wherein the sample pad contains lectin and casein or a salt thereof. 3. The lectin is a lectin having an agglutination activity of 20 μg protein / mL or less with respect to 2 volume percent of rabbit red blood cells, the immunochromatographic test strip according to 1 or 2 above. 4. The content of lectin in the specimen treatment part is 0.1 μg / cm 2 ~60 μg / cm 2 per unit area of the specimen treatment part, the immunochromatographic test strip according to 1 or 2 above. 5. The content of casein or a salt thereof in the specimen treatment part is 20 μg / cm 2 ~2,000 μg / cm 2 per unit area of the specimen treatment part, the immunochromatographic test strip according to 1 or 2 above. 6. The immunochromatographic test strip according to 2 above, wherein the blood sample is whole blood. 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 1 or 2, wherein the antibody that recognizes the component to be detected is bound to the determination section. [Effects of the Invention]

[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, supported on an impermeable substrate, and a sample processing section 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 section 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. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 shows the structure of an immunochromatographic test strip according to one embodiment of the present invention. [Figure 2] Figure 2 shows the structure of an immunochromatographic test strip according to one embodiment of the present invention. [Modes for carrying out the invention]

[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 section having a determination section on which a binding substance capable of binding to the component to be detected is immobilized, supported on an impermeable substrate, 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.

[0020] <Structure of the specimen processing area> 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, and 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-conjugated labeling reagent (antibody-conjugated labeling reagent), and more preferably that it contains an antibody-conjugated gold nanoparticle labeling reagent.

[0021] This immunochromatographic test strip consists of a sample processing section (6) for adding the test 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) for binding and separating blood cells to form plasma, and a conjugate pad (2) containing a labeling substance using an antibody that binds to a target substance in the plasma. 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 sample 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 and blood cells, the test substance is specifically bound to the 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 on the chromatograph carrier in the chromatograph medium unit (5) by capillary action, stops in the determination unit on the chromatograph medium unit (5) and is detected.

[0026] <<Sample Pad (1)>> The sample pad (1) temporarily absorbs and holds the sample. Examples of materials that make up the sample pad (1) include glass fiber, synthetic fibers such as polyacrylic fibers and polyethylene fibers, dry paper, paper pulp, textiles, regenerated cellulose fibers such as rayon and cupro, or membranes made from a combination thereof.

[0027] (Lectin) In the test strip for this immunochromatograph, the sample treatment section (6) contains lectin and casein or its salt. The content of lectin in the sample treatment section (6) is preferably 0.1 μg / cm 2 ~60 μg / cm 2 per unit area of the sample treatment section.

[0028] The content of lectin in the sample treatment section (6) is preferably 0.1 μg / cm 2 or more per unit area of the sample treatment section, 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 sample treatment section (6) [such as the sample pad (1), conjugate pad (2), blood cell separation section (3), etc.]. Therefore, blood cells will not be developed near the reaction site of at least the chromatographic medium section (5) on the downstream side of the test strip for immunochromatography, and hemolysis can be effectively suppressed in the immunochromatograph test using a blood sample as the specimen.

[0029] The content of lectin in the sample treatment section (6) is preferably 60 μg / cm 2 or less per unit area of the sample treatment section, more preferably 30 μg / cm 2 or less, and even more preferably 15 μg / cm 2 or less. When the content of the lectin is 60 μg / cm 2 or less, the effect of hemolysis suppression can be expected without unexpected non-specific reactions caused by excessive lectin within an economically feasible range.

[0030] In this immunochromatographic test strip, the lectin preferably has an agglutination activity of 40 μg protein / mL or less against 2 volume percent rabbit red blood cells. More preferably, the agglutination activity is 20 μg protein / mL or less, and even more preferably 10 μg protein / mL or less. An agglutination activity of 40 μg protein / mL or less can enhance the effect of suppressing hemolysis. The lower limit of the agglutination activity is not particularly limited, but for example, it is 2 μg protein / mL or more.

[0031] A specific method for measuring the aggregation activity of lectins includes the following steps (x1) to (x6). (x1) Place 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 procedure 3-4 times to obtain the red blood cell fraction from the stored blood. Add PBS to the red blood cell fraction obtained in (x2)(x1) to make a 2-volume percentage red blood cell suspension. (x3) Dispense 25 μL of PBS into each well of a 96-well titer plate (U-bottom), from row 2 to row 11. (x4) Place 50 μL of lectin solution into the first row of a 96-well titer plate. Take 25 μL of the lectin solution from the first row and transfer it to the well to the right (second row), then mix by pipetting. Repeat this procedure 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) The presence or absence of agglutination is determined by 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.

[0032] Examples of lectins in this embodiment include plant-derived lectins. Examples of plant-derived lectins include phytohemagglutinin (PHA), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA). Among these, PHA is preferred. Specifically, examples of PHA include lentil lectin and kidney bean lectin.

[0033] The lectin structure preferably has a carboxyl terminus, a calcium-binding site, and a sugar-binding domain, and is dimeric. Having a carboxyl terminus (C-terminus) can improve binding to sugar chains. Having a calcium-binding site can improve the structural stability of the lectin and its binding to sugar chains. Having a sugar-binding domain can show affinity for specific sugar chain patterns, potentially enhancing blood agglutination activity. Furthermore, having a dimeric structure can improve sugar chain binding and agglutination activity.

[0034] The molecular weight of the lectin varies depending on the type and origin of the lectin, but 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. A molecular weight within this range ensures sufficient blood agglutination activity.

[0035] (casein) In this immunochromatographic test strip, the casein or salt content in the sample processing area (6) is 20 μg / cm² per unit area of ​​the sample processing area. 2 ~2,000 μg / cm³ 2 It is preferable that this be the case.

[0036] The casein or salt content in the sample processing unit (6) is 20 μg / cm² per unit area of ​​the sample processing unit. 2 It is preferable that the concentration be greater than or equal to 50 μg / cm³, and more preferably 50 μg / cm³. 2 More preferably 100 μg / cm³ 2That is all. The casein or salt content is 20 μg / cm³. 2 As a result, the effect of suppressing the effects of hemolysis can be enhanced.

[0037] The casein or salt content in the sample processing unit (6) is 2,000 μg / cm² per unit area of ​​the sample processing unit. 2 Preferably, the following, and more preferably, 1,000 μg / cm³ 2 More preferably, 500 μg / cm³ 2 The following applies: The content of casein or its salt is 2,000 μg / cm³. 2 The following method makes the manufacturing process easier and more economical in terms of casein solubility. Furthermore, it can suppress problems such as delayed development of the immunochromatographic test strip due to excessive casein.

[0038] Casein is a protein extracted from dairy products, primarily existing in milk as spherical structures called micelles. Caseinate is obtained by treating casein with alkali metals (sodium, potassium) or ammonium.

[0039] 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.

[0040] 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 onto the chromatograph medium.

[0041] 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.

[0042] 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. Inclusion of mannitol can suppress hemolysis.

[0043] Examples of blocking agents include bovine serum albumin (BSA), skim milk powder, gelatin, synthetic polymers, and heat-denatured proteins.

[0044] Examples of surfactants include polyoxyethylene sorbitan monooleate 80, polyoxyethylene sorbitan monooleate 20, and octylphenol ethylene oxide additive.

[0045] Examples of buffers include phosphate buffer solution (e.g., PBS) and Tris buffer solution (e.g., TBS). Examples of protease inhibitors include phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA).

[0046] <<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 hinder the resolubility of the labeled detection reagent in the developing solution when a sample containing the substance to be detected is developed during testing.

[0047] <<Blood cell separation section (3)>> The blood cell separation section (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 section (3) include synthetic polymer fibers such as cellulose and polyamide, and membranes such as thin filter paper made of glass fiber fibers.

[0048] The membrane only needs to be capable of separating red blood cells contained in the whole blood sample, and preferably it also has the function of preferentially spreading plasma components onto the membrane by separating other blood cell components such as white blood cells and platelets, or by drastically reducing their spread rate.

[0049] 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.

[0050] <Chromatography media section (5)> The chromatographic medium portion (5) used in this immunochromatographic test strip has a determination portion supported on an impermeable substrate, on which a binding substance capable of binding 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 portion.

[0051] 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 substance 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.

[0052] In this embodiment, the chromatographic medium portion (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 cellulose derivatives 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.

[0053] 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.

[0054] 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 made of plastic or the like on the back surface of the chromatographic medium (5) on which the reaction site is formed. The properties of this support are not particularly limited, but when observing the measurement results by visual inspection, it is preferable that the support has a color that does not resemble the color produced by the labeling substance, and is usually colorless or white.

[0055] <Judgment part> In this immunochromatographic test strip, a detection area (reaction site) is formed on the chromatographic medium (5) used, on which a substance that specifically binds to the substance to be detected, such as an antibody, is immobilized as an immobilization reagent at an arbitrary position. 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 fine particles such as latex particles, and these fine particles are captured and immobilized on the chromatographic medium (5).

[0056] Methods for direct immobilization include physical adsorption and covalent bonding. Generally, when the chromatographic medium is a (5) nitrocellulose membrane or a mixed nitrocellulose ester membrane, physical adsorption can be performed.

[0057] 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 method of immobilization, an immobilization reagent is bound to insoluble microparticles, and then immobilized on the chromatographic medium (5).

[0058] 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 about 10 μm or less. Various types of particles used in antigen-antibody reactions are known and these known particles can also be used in this embodiment.

[0059] 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-methacrylic acid copolymer, polyglycidyl methacrylate, and acrolein-ethylene glycol dimethacrylate copolymer; microparticles of gelatin, bentonite, agarose, and crosslinked dextran; and inorganic oxides such as silica, silica-alumina, and alumina, as well as inorganic particles into which functional groups have been introduced by silane coupling treatment or other methods.

[0060] 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 extending perpendicular to the development direction of the chromatographic medium (5), numbers, letters, or symbols such as + and -.

[0061] 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.

[0062] In addition, the chromatograph medium section (5) may include, as needed, a sample addition section (sample pad, etc.) for adding a sample containing the substance to be detected, a section for removing solid components such as blood cells from the sample (blood cell separation section, etc.), a developing solution addition section for adding developing solution, an absorption section (absorption pad, etc.) for absorbing the substance to be detected and developing solution that were not captured by the reaction section, and a control section to indicate that the measurement was performed correctly. The materials of these sections 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 capillaries to the chromatograph medium section (5) on which the immobilized reagents are immobilized.

[0063] <Labeling substance> The detection reagent used in this embodiment is a substance that specifically binds to the substance to be detected, such as an antibody, and is labeled with a labeling substance. While enzymes are generally used to label detection reagents in immunochromatography, an insoluble carrier is used as the labeling substance in this embodiment because it is suitable for visually determining the presence of the substance to be detected. In this embodiment, a labeled detection reagent is prepared by sensitizing the detection reagent to an insoluble carrier.

[0064] 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. In particular, colloidal gold particles are preferred because they allow for easy detection.

[0065] Insoluble carriers are suitable labeling materials for visually determining the presence of a substance 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.

[0066] Since latex particles made of synthetic polymers are naturally white, they cannot be used as labeling substances as they are. However, by dyeing them with oil-soluble dyes, particularly latex particles in an aqueous medium, using an emulsion of an oil-soluble dye solution in an oily organic solvent, they can be made to have a desired color and intensity.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] With respect to these insoluble carriers, it is known that the surface of both latex particles and colloidal metal particles is 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).

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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 is prepared by adding antibody to a solution in which gold particles are dispersed colloidally and allowing physical adsorption, and then adding bovine serum albumin solution to block the surface of particles to which the antibody is not bound.

[0075] 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 development path of the mobile phase 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.

[0076] 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.

[0077] 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 capillaries.

[0078] <Detected substances> The substances to be 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 portion of glycoproteins, the sugar portion of glycolipids, etc.), and complex carbohydrates.

[0079] In this specification, "specifically binding" means binding based on the affinity between biomolecules. Examples of such affinity-based binding include the binding of antigens to antibodies, sugars to lectins, hormones to receptors, enzymes to inhibitors, complementary nucleic acids to each other, and nucleic acids to nucleic acid-binding proteins. Therefore, when the substance to be detected is antigenic, examples of substances that specifically bind to the substance to be detected include polyclonal antibodies or monoclonal antibodies. Also, when the substance to be detected is a sugar, examples of substances that specifically bind to the substance to be detected include lectin proteins.

[0080] Specific substances that can 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 beta-hemolytic streptococcus antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, and glycated albumin.

[0081] The immunochromatographic test strip, consisting of the above components, can be used as is, or, if necessary, placed in a molded plastic housing.

[0082] <Developing solution> The developing solvent used in this embodiment is a liquid that constitutes the mobile phase in immunochromatography, and moves along the chromatographic medium, which is the stationary phase, together with the sample containing the substance to be detected and the labeled detection reagent. Any such developing solvent may be used.

[0083] As described above, the following configurations are disclosed in this specification. 1. A chromatographic medium section having a determination section on which a binding substance capable of binding to the component to be detected is immobilized, supported on an impermeable substrate, An immunochromatographic test strip, comprising a sample processing section to which a sample is added, is supported on an impermeable substrate at a position upstream of the chromatographic medium section in the sample development direction, An immunochromatographic test strip in which the sample processing section contains lectin and casein or a salt thereof. 2. The sample processing unit consists of a sample pad to which a blood sample is added, a blood cell separation unit to which blood cells are separated, and a conjugate pad containing a labeling substance. The immunochromatographic test strip according to claim 1, wherein the sample pad contains 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 / cm² per unit area of ​​the sample processing unit. 2 ~60 μg / cm³ 2 The immunochromatographic test strip according to any one of the above 1 to 3. 5. The casein or salt content in the sample processing unit shall be 20 μg / cm² per unit area of ​​the sample processing unit. 2 ~2,000 μg / cm³ 2 The immunochromatographic test strip described in any one of the above 1 to 4. 6. An immunochromatographic test strip according to any one of items 2 to 5 above, wherein the blood sample is whole blood. 7. An immunochromatographic test strip according to any one of 1 to 6, 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 antibody that recognizes the component to be detected is bound to the determination portion.

[0084] The present invention will be described below based on examples, but the present invention is not limited thereto. [Examples]

[0085] 1. Preparation of test strips for immunochromatography (1) Preparation of chromatography medium A nitrocellulose sheet, HF240 (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 a carbonate buffer (pH 9.0) containing 5% by mass of isopropyl alcohol. 40 μL of this solution was spread onto the membrane in a 1 mm width and dried overnight at 60°C to prepare the chromatographic medium.

[0086] (2) Preparation of labeled substance solution To 0.5 mL of a gold colloid dispersion (manufactured by Tanaka Kikinzoku Kogyo Co., Ltd.: LC40nm), 0.1 mL of anti-PSA monoclonal antibody (secondary antibody), diluted to a concentration of 0.025 mg / mL with Bicine 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 thorough stirring, the mixture was 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.

[0087] (3) Fabrication of conjugate pads 300 μL of the labeling reagent solution prepared above was mixed with 300 μL of 10 wt% 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 a conjugate pad.

[0088] (4) Preparation of sample pads Using a 16mm x 300mm fiberglass pad (SureWick, trade name, Millipore), 2.4 mL of 50 mM phosphate buffer (pH 8.0) containing 1% by mass of Tween® 80, 5% by mass of mannitol, and 0.0075 mg / mL of SSA, ABA, PHA-E4 (MGC Corporation) or 0.5% by mass of sodium caseinate (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 a sample pad.

[0089] (5) Blood cell separation section A GF / DVA (product name, manufactured by Whatman: 180mm x 30mm) was used as the blood cell separation unit.

[0090] (6) Preparation of test strips for immunochromatography The chromatograph 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 were attached to a base material consisting of a backing sheet coated with adhesive on one side. Then, the strips were cut with a cutting machine to a width of 5 mm to form immunochromatographic test strips.

[0091] 2. Immunochromatographic measurement (detection of PSA in blood using human whole blood) PSA detection in blood was performed 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 sample was placed on the sample pad of the immunochromatographic test strip and allowed to unfold. Visual determination was made after 30 minutes. In Table 1, PSA is represented as "antigen".

[0092] PSA is detectable in very small amounts even in healthy individuals, and the normal range is generally 4 ng / mL. A level of 10 ng / mL is considered to indicate a 50% or higher 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.

[0093] In the presence of the antigen, negative and positive samples were used, and each condition was tested three times. Visual evaluation was performed, and the number of times hemolysis occurred out of the three trials was counted as the "number of times hemolysis was observed." An overall evaluation was also made according to the following criteria. The results are shown in Table 1. (In the case of a negative sample with a PSA concentration of 1 ng / mL in the blood) A1: No hemolysis was observed, and no color change was seen in the test line. B1: There is some hemolysis, but no color development is observed in the test line. C1: Hemolysis is present, and the test line cannot be identified. (In the case of a positive sample with a PSA concentration of 30 ng / mL) A2: No hemolysis was observed, and the red line on the test line was visible. B2: There is some hemolysis, but the red line on the test line is visible. C2: Hemolysis is present, and a red line can be seen on the test line.

[0094] The reagents shown in Table 1 are described below. SSA: Japanese elderberry collectin (aggregation activity <20 μg protein / mL) manufactured by MGC Corporation. ABA: Mushroom lectin manufactured by MGC, aggregation activity <20 μg protein / mL PHA-E4: Kidney bean lectin-E4 manufactured by MGC, agglutination activity <10 μg protein / mL

[0095] [Table 1]

[0096] 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 did not contain lectin and a salt of casein experienced hemolysis, making it difficult to distinguish the color development of the detection area, resulting in a decrease in detection sensitivity. [Explanation of symbols]

[0097] 1. Sample Pad 2. Conjugate pad 3. Blood cell separation section 4. Packing sheet 5. Chromatography media section 6. Specimen Processing Section 7. Absorbent pads

Claims

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

2. The sample processing unit consists of a sample pad to which a blood sample is added, a blood cell separation unit to which blood cells are separated, and a conjugate pad containing a labeling substance. The immunochromatographic test strip according to claim 1, wherein 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 whole blood.

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 antibody that recognizes the component to be detected is bound to the determination portion.

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