WO2020230826A1 - Tampon absorbant pour kit de diagnostic immunochromatographique et kit de diagnostic immunochromatographique - Google Patents

Tampon absorbant pour kit de diagnostic immunochromatographique et kit de diagnostic immunochromatographique Download PDF

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Publication number
WO2020230826A1
WO2020230826A1 PCT/JP2020/019144 JP2020019144W WO2020230826A1 WO 2020230826 A1 WO2020230826 A1 WO 2020230826A1 JP 2020019144 W JP2020019144 W JP 2020019144W WO 2020230826 A1 WO2020230826 A1 WO 2020230826A1
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Prior art keywords
pad
diagnostic kit
immunochromatographic
absorption pad
immunochromatographic diagnostic
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PCT/JP2020/019144
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English (en)
Japanese (ja)
Inventor
厚志 堀井
武志 松瀬
佐藤　潤一
雄一 原
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旭化成株式会社
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Priority to CN202080030364.8A priority Critical patent/CN113767287A/zh
Priority to JP2021519465A priority patent/JPWO2020230826A1/ja
Priority to KR1020217023217A priority patent/KR20210107781A/ko
Publication of WO2020230826A1 publication Critical patent/WO2020230826A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to an absorption pad for an immunochromatographic diagnostic kit. More specifically, the present invention relates to an absorption pad for an immunochromatographic diagnostic kit that has excellent reproducibility of test results and does not cause regurgitation.
  • a sandwich method As the measurement principle of the immunochromatography method, there are a method called a sandwich method and a method called a competitive method. Further, as a measurement format, there are methods called a flow-through type and a lateral flow type.
  • Various substances can be detected as the substances to be tested in the sample, and a typical example is the measurement of detecting the antigen by the sandwich method, and the following operations are sequentially executed.
  • An antibody that specifically binds to an antigen, which is a substance to be tested is immobilized on a predetermined site of a chromatographic medium such as a chromatographic medium membrane, and a test line (hereinafter referred to as “TL”) is placed at an arbitrary position on the chromatographic medium.
  • TL test line
  • a detection reagent in which an antibody that specifically binds to the substance to be inspected is carried on a labeling substance such as an enzyme, a labeling substance, a fluorescent labeling substance, or magnetic particles, and apply the detection reagent to a conjugate pad or the like to dry. Then, a detection reagent-containing portion is formed, and the immunochromatographic diagnostic kit is formed by combining with the chromatograph medium. (3) The sample itself containing the antigen or a solution obtained by diluting it with an arbitrary liquid is dropped at a predetermined position of the immunochromatographic diagnostic kit, for example, on a sample pad, and the antigen and the detection reagent are developed on a chromatograph medium. .. By these operations, the labeling substance is captured by the antibody immobilized on the chromatograph medium at the reaction site via the antigen, and the signal of the labeling substance is detected to make a diagnosis by the immunochromatographic diagnostic kit.
  • a labeling substance such as an enzyme, a labeling substance, a fluorescent labeling
  • the cause of the variation is considered to be the influence of each member that composes the kit, but since the absorption pad is the part that absorbs the liquid, it causes the variation in the color development intensity due to defects such as background coloring. ..
  • the “background” is a phenomenon that occurs when the labeling reagent is clogged in the pores of the chromatographic medium film or non-specifically adsorbed, so that the chromatographic medium film is colored. If the background is poor, the contrast between the chromatograph medium film and the test line (TL) will be poor, making it difficult to see the colored lines of the TL, making it difficult to make a judgment in the medical field. Further, if the background is bad, it becomes difficult to judge a negative TL (that is, no color development). These phenomena can lead to misdiagnosis.
  • background coloring due to backflow of labeling reagents after judgment.
  • the determination time is set for each immunochromatographic diagnostic kit, but in a clinical site where the patient is extremely busy, it is often impossible to make a determination exactly at the determination time.
  • backflow of labeling reagent means that the labeling reagent once absorbed by the absorption pad flows back due to the drying of the chromatograph medium and develops in the opposite direction on the chromatograph medium. This backflow can cause misdiagnosis by coloring the background and even polluting the TL.
  • an object to be solved by the present invention is to provide an absorption pad for an immunochromatographic diagnostic kit that has excellent reproducibility of test results and does not cause reflux.
  • the present invention is as follows.
  • the immunochromatographic diagnostic kit using the absorption pad for the immunochromatographic diagnostic kit of the present invention has excellent reproducibility of test results and does not cause regurgitation.
  • the absorption pad for an immunochromatographic diagnostic kit of the present embodiment is an absorption pad for an immunochromatographic diagnostic kit containing carboxymethylated cellulose fibers, and is an average substitution of hydroxyl groups in glucose units constituting the carboxymethylated cellulose fibers.
  • the degree is 0.05 or more and 1.5 or less.
  • the immunochromatographic test method performed using an immunochromatographic diagnostic kit generally includes a flow-through method in which a solution containing an object to be measured is passed in a direction perpendicular to a membrane and a lateral flow method in which a solution containing an object to be measured is passed in a horizontal direction.
  • the absorption pad for the immunochromatographic diagnostic kit of the present embodiment can be used in any of these methods, and is not limited to these in the sense of the system of the immunochromatographic diagnostic kit that absorbs water.
  • the carboxymethylated cellulose fiber contained in the absorption pad for the immunochromatographic diagnostic kit of the present embodiment has an average degree of substitution of hydroxyl groups in the glucose unit constituting the cellulose when carboxymethylated is 0.05 or more and 1.5 or less. Is. If the average degree of substitution is too high, a water block will be created in the front part of the absorption pad when the developed liquid is absorbed. As a result, a sufficient amount of the developing liquid is not absorbed, and further, development failure occurs, resulting in inspection variation.
  • the upper limit of the average degree of substitution is preferably 1.3 or less, more preferably 0.9 or less. More preferably, it is 0.6 or less. If the average degree of substitution is 0.05 or more, a sufficient amount of liquid absorption can be secured.
  • the lower limit of the average degree of substitution is preferably 0.1 or more, and more preferably 0.2 or more.
  • the terminal of the carboxymethyl group is a sodium salt, but after that, it is treated with an acid to convert some carboxymethyl groups into a proton type. can do.
  • the method for partially protonating the carboxymethylated cellulose fiber is not particularly limited, but the protonation is carried out by immersing the carboxymethylated cellulose fiber in acetic acid, hydrochloric acid, or nitric acid adjusted to a predetermined concentration using a solvent containing alcohol. It is more preferable that the protonation is carried out by immersing the mixture in acetic acid adjusted to a predetermined concentration using a solvent containing alcohol.
  • the absorption pad for an immunochromatographic diagnostic kit containing the carboxymethylated cellulose fiber of the present embodiment preferably has a catch and release index of 0.5 or less.
  • the "catch and release index” is an index that indicates the balance between the property of the absorbing pad to absorb the applied liquid and the property of the absorbing pad to hold the applied liquid.
  • Catch and release index (100 x C ⁇ D) ⁇ T ⁇ In the formula, C: liquid absorption amount (g / 100 cm 2 ), D: basis weight (g / m 2 ), T: release time (minutes) ⁇ . If the catch-and-release index is too large, the labeling reagent once absorbed by the absorption pad may flow back on the chromatograph medium or test variation may occur.
  • the upper limit of the catch and release index is more preferably 0.3 or less, still more preferably 0.1 or less.
  • the lower limit is preferably 0.0001 or more, and more preferably 0.001 or more.
  • the catch-and-release index is determined by controlling the average substitution degree, average fiber diameter, fiber length, etc. of the carboxymethylated cellulose fiber, and / or the morphology of the absorption pad containing the carboxymethylated cellulose fiber. Can be adjusted.
  • the term "release time” means that an absorption pad having a width of 4 mm and a length of 25 mm is placed on a chromatographic medium having a width of 4 mm and a length of 25 mm without deviation, and 20 ⁇ L of physiological saline is applied. It is a numerical value in minutes that indicates the time required for the saline solution to start migrating from the absorption pad to the chromatographic medium when it is gently and evenly dropped onto the absorption pad. For the measurement of "release time”, if the relationship between the absorption pad size and the amount of physiological saline is the same as the above method, the absorption pad size and physiological saline are different from the above method. The amount of water may be used.
  • the average fiber diameter of the carboxymethylated cellulose fibers is preferably 1 ⁇ m or more and 100 ⁇ m or less. If the average fiber diameter is too large, a water block will be created in the front part of the absorption pad when the developed liquid is absorbed. As a result, a sufficient amount of the developing liquid is not absorbed, and further, development failure occurs, resulting in inspection variation.
  • the upper limit of the fiber diameter is preferably 80 ⁇ m or less, and more preferably 70 ⁇ m or less. If the average fiber diameter is 1 ⁇ m or more, a sufficient amount of liquid absorption can be secured.
  • the lower limit of the average fiber diameter is preferably 3 ⁇ m or more, and more preferably 5 ⁇ m or more.
  • the absorption pad for the immunochromatographic diagnostic agent kit containing the carboxymethylated cellulose fibers of the present embodiment can be in the form of a woven fabric, a knitted fabric, or a non-woven fabric.
  • G / m 2 that is, the weight of the non-woven fabric (g) / the area of the non-woven fabric (m 2 )
  • the lower limit of the basis weight is more preferably 20 g / m 2 or more, and further preferably 30 g / m 2 or more.
  • the upper limit of the basis weight is more preferably 350 g / m 2 or less, further preferably 300 g / m 2 or less, still more preferably 250 g / m 2 or less, still more preferably 200 g / m 2 or less, still more preferably 190 g / m. It is 2 or less, more preferably 180 g / m 2 or less.
  • the thickness of the absorption pad for the immunochromatographic diagnostic kit containing the carboxymethylated cellulose fiber of the present embodiment is preferably 0.03 mm or more and 10.00 mm or less in the case of a woven fabric or a sheet, for example. If the pad is too thick, for example, when used in an immunochromatographic diagnostic kit, it becomes difficult to store the pad in a housing, and as a result, the diagnostic results vary widely.
  • the upper limit of the thickness of the pad is more preferably 9.00 mm or less, still more preferably 8.00 mm or less. On the other hand, if the sheet is too thin, a sufficient amount of liquid absorption cannot be secured as an absorption pad for the immunochromatographic diagnostic kit.
  • the lower limit of the thickness of the pad is more preferably 0.05 mm or more, still more preferably 0.10 mm or more.
  • the "thickness" of the pad means a value obtained by measuring a load of 1.96 kPa in a thickness test conforming to JIS-L1096.
  • the absorption pad for the immunochromatographic diagnostic kit of the present embodiment preferably has a liquid absorption amount of 5 g / 100 cm 2 or more and 50 g / 100 cm 2 or less when impregnated with physiological saline. If the amount of liquid absorbed is too large, the structure will collapse when the liquid is absorbed when used in an immunochromatographic diagnostic kit, resulting in poor deployment.
  • the upper limit of the amount of liquid absorbed is preferably 50 g / 100 cm 2 or less, and more preferably 30 g / 100 cm 2 or less. If the amount of liquid absorbed is less than 5 g / 100 cm 2 , the desired effect may not be exhibited and backflow may occur.
  • the lower limit of the amount of liquid absorbed is preferably 5 g / 100 cm 2 or more, and more preferably 10 g / 100 cm 2 or more.
  • the cellulose fiber (before carboxymethylation) that is the raw material of the carboxymethylated cellulose fiber is not particularly limited, and is known as a known cellulose fiber such as cuprammonium rayon, viscose rayon, lyocell, cotton, pulp, and polynosic. , Preferred are cuprammonium rayon, viscose rayon, and more preferably cuprammonium rayon.
  • Some cellulose fiber materials have a low degree of polymerization, and in such materials, if the degree of substitution is high, the fibers are easily decomposed and it is necessary to suppress the degree of substitution to the lowest possible, whereas when cuprammonium rayon is used, It has the advantages of high degree of polymerization and resistance to disintegration even at a relatively high degree of substitution, and a wide range of degree of substitution can be selected.
  • the cellulose fiber may be either a long fiber or a short fiber.
  • the long fibers continuous long fibers are preferable.
  • the long fiber means a fiber having a fiber length of 10 mm or more, and the fiber length is preferably 20 mm or more, more preferably 50 mm or more, and further preferably a continuous long fiber. Since the short fiber cellulose sheet is a short fiber, if the degree of substitution is high, the fibers are easily separated and it is necessary to suppress the degree of substitution as low as possible, whereas when the continuous long fiber sheet is used, the degree of substitution is relatively high. It has the advantage of being hard to collapse and it is possible to select a wide range of substitution degrees.
  • the absorption pad for the immunochromatographic diagnostic kit containing the carboxymethylated cellulose fibers of the present embodiment is preferably in the form of cotton, woven fabric, knitted fabric or non-woven fabric, and more preferably carboxymethylated. It is in the form of a woven fabric or non-woven fabric of regenerated cellulose fibers, more preferably a non-woven fabric of carboxymethylated regenerated cellulose fibers. In the non-woven fabric form, the fibers are entangled even in the gelled state when wet, so that high liquid absorption can be obtained while maintaining the strength when wet, which is suitable as an absorption pad for an immunochromatographic diagnostic kit. Can be used for.
  • the absorbent pad for immunochromatographic diagnostic kit containing the carboxymethylated cellulose fiber of the present embodiment includes fibers other than those derived from natural or regenerated cellulose fiber, for example, polyester fiber, polypropylene fiber, nylon, as long as the desired action and effect are not impaired.
  • Synthetic fibers such as fibers and bioabsorbable fibers such as polyglycolic acid, polylactic acid, and polyapple acid may be contained.
  • such a material is preferably 50% or less, and more preferably 30% or less.
  • Such fibers may be long fibers or short fibers. As the long fibers, continuous long fibers are preferable.
  • the absorption pad for the immunochromatographic diagnostic kit of the present embodiment may be a stack of sheets depending on various uses.
  • heat embossing heat embossing
  • heat fusion methods such as ultrasonic fusion
  • mechanical entanglement methods such as needle punching and water jet
  • hot melt adhesives urethane adhesives and other adhesive methods
  • extrusion hot melt adhesives
  • urethane adhesives and other adhesive methods and extrusion.
  • Various known methods such as laminating can be adopted, and the present invention is not particularly limited.
  • the sheet material used for laminating the sheets is not limited in any way, and may be a bioabsorbable polymer such as cellulose, a thermoplastic resin, or polyglycolic acid, polylactic acid, or polyapple acid.
  • the thermoplastic resin can be a polyolefin-based resin, a polyester-based resin, or a polyamide-based resin, and specifically, ethylene, propylene, 1-butene, 1-hexene, 4-methyl-1-.
  • High-pressure low-density polyethylene linear low-density polyethylene (LLDPE), high-density polyethylene, polypropylene (propylene homopolymer), polypropylene random copolymer, which is a single or copolymer of ⁇ -olefins such as penten and 1-octene.
  • LLDPE linear low-density polyethylene
  • HPPE high-density polyethylene
  • polypropylene propylene homopolymer
  • polypropylene random copolymer which is a single or copolymer of ⁇ -olefins such as penten and 1-octene.
  • any one or a mixture of polyethylene terephthalate, polybutylene terephthalate, polypropylene, and polyethylene is suitable from the viewpoint of stability against heat and moisture and versatility.
  • thermoplastic resins are hydrophobic, and even if the structure is formed as a non-woven fabric, there is almost no one that absorbs water.
  • the variation in water absorption can be reduced by using a non-woven fabric composed of hydrophobic thermoplastic resin fibers and performing a hydrophilic treatment.
  • the method of hydrophilization is not particularly limited, and if it is a physical processing method, for example, hydrophilization by corona treatment or plasma treatment can be mentioned, and a chemical processing method, for example, introduction of a surface functional group, For example, a sulfonic acid group, a carboxylic acid group, or the like can be introduced by an oxidation treatment or the like, or a water-soluble polymer such as polyvinyl alcohol (PVA), a polyester resin, polystyrene sulfonic acid, or polyglutamic acid, and / or a surfactant.
  • PVA polyvinyl alcohol
  • a treatment agent such as a nonionic surfactant, an anionic surfactant, a cationic surfactant, or an amphoteric surfactant is impregnated and coated on a non-woven fabric by a dip nip method or a spray coating method.
  • a nonionic surfactant such as a nonionic surfactant, an anionic surfactant, a cationic surfactant, or an amphoteric surfactant
  • examples thereof include known methods such as a method for In consideration of the water absorption required for the absorption pad, an appropriate hydrophilic processing method and conditions, for example, the amount of the treatment agent used and the amount of the functional group introduced can be selected.
  • the method for producing the non-woven fabric is not particularly limited, and examples thereof include spunbonded non-woven fabric, melt-blown non-woven fabric, wet non-woven fabric, dry non-woven fabric, dry pulp non-woven fabric, flash-spun non-woven fabric, and spread non-woven fabric.
  • the non-woven fabric made of ultrafine fibers melt-blown non-woven fabric and flash-spun non-woven fabric are preferable.
  • non-woven fabric used for laminating the sheets those having a basis weight of 20 g / m 2 or more and 200 g / m 2 or less, a thickness of 0.06 mm or more and 1.20 mm or less, and an average fiber diameter of 0.7 ⁇ m or more and 5.0 ⁇ m or less are preferable. Can be used for.
  • An example of a method for producing an absorption pad for an immunochromatographic diagnostic kit containing the carboxymethylated cellulose fiber of the present embodiment is shown below, but the method is not limited to this method.
  • the structure of the natural or regenerated cellulose fiber is stirred at 35 ° C. for 30 minutes while maintaining an alkaline state in an aqueous alcohol-containing sodium hydroxide solution.
  • sodium monochloroacetate containing alcohol is added, and the mixture is stirred at 30 ° C. to 55 ° C. for 1 to 12 hours.
  • the degree of substitution is controlled by the bath ratio of the reaction solution and the structure, the temperature, and the time.
  • reaction conditions can be appropriately changed in consideration of production cost and the like.
  • the obtained structure is adjusted to pH 6.0 to 8.0 with an aqueous acetic acid-containing ethanol solution, and then alcohol is replaced with 70 wt%, 90 wt%, or 100 wt% ethanol. If it contains even a small amount of water, it becomes hard, so by gradually increasing the alcohol concentration, alcohol replacement can be reliably performed and morphological stability can be maintained. Then, the mixture is immersed in an acid-containing ethanol solution adjusted to a predetermined concentration, stirred for 1 hour, alcohol-substituted with 70 wt%, 90 wt%, and 100 wt% ethanol, and dried to obtain a protonated sheet-like structure.
  • the mixture is immersed in an aqueous ethanol solution containing calcium hydroxide adjusted to a predetermined concentration, stirred for 1 hour, alcohol-substituted with 70 wt%, 90 wt%, and 100 wt% ethanol, and dried to obtain a sheet-shaped pad according to the embodiment of the present application. ..
  • the "diagnostic method” performed using the immunochromatographic diagnosis kit using the absorption pad for the immunochromatographic diagnosis kit of the present embodiment refers to various diagnoses performed using the immunochromatographic diagnosis kit in vitro.
  • the diagnostic target is not particularly limited, and can be used for inspections of various diagnostic targets such as human, animal, food, plant, and other environmental tests.
  • a sample sample is collected from the test target, if necessary, pretreated such as extraction and filtration, dropped onto the sample pad, wait for a predetermined time from the start of the test, and the substance to be tested The diagnosis result is judged from the color development that differs depending on the presence or absence.
  • the procedure is not limited to this, and can be used for diagnosis of similar procedures and principles. It is preferable that the sample sample is filtered in advance to remove excess foreign substances and impurities, which can be expected to further speed up the diagnosis and improve the diagnostic accuracy.
  • the immunochromatographic diagnostic kit of the present embodiment is for easily detecting the presence or absence of a substance to be tested in various samples.
  • Types of diagnostic kits include lateral flow type and flow-through type. The type is not particularly limited as long as it uses a labeling reagent or an absorption pad, but is preferably a lateral flow type. Further, among the lateral flow types, there are a dipstick type and a cassette type, but these types are not particularly limited.
  • the configuration of the diagnostic kit is not particularly limited, and any configuration generally used in the art may be used.
  • the member is not particularly limited as long as it is used in the art, and for example, (a) sample pad, (b) conjugate pad (including antibody sensitization labeling reagent), and (e) chromatograph shown in FIG.
  • Examples include a graph medium film, (f) an absorbent pad, and (g) a mount. In addition, some of these members may be omitted if necessary.
  • the absorption pad is the part that finally absorbs the sample to be measured in immunochromatography
  • (c) is the test line (TL)
  • (d) is the control line ( CT).
  • examples of the general absorption pad of the prior art include cellulose filter paper, paper, glass fiber, and glass fiber.
  • the sample pad is the part that first receives the sample to be measured in immunochromatography.
  • sample pads include cellulose filter paper, paper, glass fiber, glass fiber, acrylic fiber, nylon fiber, and various woven fabrics.
  • pretreatment may be performed as necessary.
  • a buffer solution, a surfactant, a protein, a reagent for trapping impurities in a sample sample, a preservative, an antibacterial agent, an antioxidant, a hygroscopic agent, and the like may be contained in advance.
  • the conjugate pad is a part where labeled particles such as an antibody-sensitized labeling reagent are dried and immobilized.
  • labeled particles such as an antibody-sensitized labeling reagent are dried and immobilized.
  • general conjugate pads include glass fiber, glass fiber, acrylic fiber, PET fiber alone or composite non-woven fabric, and woven fabric.
  • pretreatment may be performed as necessary.
  • the labeling reagent fixed to the conjugate pad refers to a particulate matter that is insoluble in water, buffer solution, etc. and carries a dye, dye, etc.
  • the material constituting the particles is not particularly limited, and examples of such labeling reagents include metal colloidal particles such as gold colloid, platinum colloid, silver colloid, and selenium colloid, and styrene-based latex such as polystyrene latex and acrylic acid-based latex. Colored latex particles colored with, etc., colored silica particles colored silica composed of a three-dimensional structure composed of silicon atoms and oxygen atoms, colored cellulose particles colored with cellulose, labeling reagents in which colored components such as carbon black are directly granulated. , Magnetic particles, and the like. Further, the labeling reagent may be fluorescent luminescent particles.
  • the labeling reagent needs to support a substance that specifically binds to the substance to be detected, such as an antibody, but the method of supporting the substance is not particularly limited. For example, support by physical adsorption, support by covalent bond, support by a combination thereof, and the like can be mentioned. The type and amount of the substance to be carried are not particularly limited. Antibodies are the most common and preferable types of substances to be carried. Further, as the method of supporting, the support by physical adsorption is preferable from the viewpoint of ease, and the support by covalent bond is preferable from the viewpoint of stability and performance.
  • the target that can be diagnosed by the immunochromatographic diagnostic kit of the present embodiment is not particularly limited, but specific examples include the following: cancer markers, hormones, infectious diseases, autoimmunity, blood proteins, TDM, coagulation. ⁇ Fibrinolysis, amino acids, peptides, proteins, genes, cells, etc. More specifically, CEA, AFP, ferritillin, ⁇ 2 micro, PSA, CA19-9, CA125, BFP, elastase 1, pepsinogen 1.2, fecal occult blood, urinary ⁇ 2 micro, PIVKA-2, urinary BTA, insulin.
  • the chromatographic medium used in the immunochromatographic diagnostic kit is not particularly limited, and various commonly used chromatographic media can be used. Specific examples include a nitrocellulose membrane.
  • a dispersion of a labeling reagent adjusted to a predetermined concentration is prepared, a buffer solution and an antibody are added, and the mixture is stirred for a certain period of time while adjusting the temperature to adsorb the antibody to the labeling reagent.
  • the colored cellulose particles are blocked by further adding a blocking agent and stirring for a certain period of time while adjusting the temperature.
  • a blocking agent various blocking agents can be used depending on the composition of the substance to be tested, the sample, or the solution diluting the sample.
  • centrifugation is performed to separate the supernatant containing excess antibody and blocking agent from the precipitated particles, and the supernatant is removed by decantation.
  • a liquid such as a buffer solution is added to the precipitated particles, and if necessary, dispersion treatment is performed by ultrasonic waves or the like. Washing is performed a required number of times by a series of operations such as sedimentation by centrifugation, removal of supernatant, and addition of liquid to prepare a dispersion containing particles having antibody adsorption / blocking at a predetermined concentration.
  • a buffer solution, a surfactant, a protein, a reagent for trapping impurities in a sample sample, an antiseptic, an antibacterial agent, an antioxidant, a hygroscopic agent, etc. are applied to the regenerated cellulose continuous long fiber non-woven fabric as necessary, and dried. And prepare a sample pad.
  • a chromatographic medium made of a chromatographic medium film in which an antibody is immobilized at a predetermined position, and an absorption pad made of cellulose filter paper for absorbing a sample are prepared.
  • An immunochromatographic diagnostic kit is produced by immobilizing them on a sheet having an adhesive site called a backing sheet and cutting them into a predetermined size.
  • Equation (1) Calculated by ⁇ in the formula, f: 0.1 mol / L potassium hydroxide titer ⁇ .
  • the alkalinity is read as the acidity.
  • (Ii) Average Substitution Degree Weigh 0.5 to 0.7 g of a sample (anhydride) precisely, wrap it in filter paper, and incinerate it in a magnetic crucible. After cooling, transfer this to a 500 mL beaker, add about 250 mL of water and 35 mL of 0.05 mol / L sulfuric acid with a pipette, and boil for 30 minutes.
  • Equation (3) ⁇ In the formula, A: amount of 0.05 mol / L sulfuric acid consumed in 1 g of sample (mL), a: amount of 0.05 mol / L sulfuric acid used (mL), f: 0.05
  • the degree of substitution was calculated from the molar / L sulfuric acid titer, b: 0.1 molar / L potassium hydroxide titration (mL), f1: 0.1 molar / L potassium hydroxide titer ⁇ , and the average thereof.
  • N 3 or more
  • Liquid absorption C (g / 100 cm 2 ) (BA) x 4. .. .. Equation (4) ⁇ In the formula, A: weight in a dry state before immersion (g), B: weight after incubation for 30 minutes (g) ⁇ , the amount of liquid absorbed is calculated.
  • ⁇ Metsuke D (g / m 2 )> A non-woven fabric having an area of 0.5 m 2 or more is dried at 105 ° C. until it reaches a constant weight, and then left in a constant temperature room at 20 ° C. and 65% RH for 16 hours or more to measure the weight, and per unit area of the non-woven fabric. The weight was measured.
  • ⁇ Thickness> A value obtained by measuring a load of 1.96 kPa in a thickness test based on JIS-L1096.
  • ⁇ Release time (minutes)> Prepare a plurality of sets of test pieces in which absorption pads having a width of 4 mm and a length of 25 mm are stacked on a chromatographic medium made of nitrocellulose having a width of 4 mm and a length of 25 mm so as not to shift, and each test piece is prepared with physiological saline. 20 ⁇ L of each was gently and evenly dropped onto the absorption pad of. When time T1 (minutes) had elapsed from the completion of dropping the physiological saline, the absorption pad was removed from the chromatographic medium for one set of test pieces, and the surface condition of the chromatographic medium was visually observed.
  • the release time of the absorption pad was set to T1 (minutes).
  • T1 minutes
  • T2 minutes
  • T1 ⁇ T2 time T1 ⁇ T2
  • the release time was determined by repeating the above operation until a color change due to wetting was observed on the surface of the chromatographic medium. However, if no change in the color of the surface of the chromatographic medium due to liquid wetting is observed in any time from the completion of dropping the saline solution to the elapse of 900 minutes, the release time of the absorption pad is 900. It was a minute.
  • BG coloring ⁇ Background of immunochromatographic diagnostic kit (BG coloring)> An immunochromatographic kit cut to the appropriate width was placed in a plastic housing. Next, a 66 mM PBS containing 1 wt% BSA and a pH of 7.4 was prepared and used as a negative sample. Using the obtained diagnostic kit in a housing, 100 ⁇ L of a negative sample was dropped onto the sample dropping part of the diagnostic kit, and 5 minutes later, using an immunochromatographic reader C10066-10 manufactured by Hamamatsu Photonics, 1 between TL and CL. The color development intensity of the points (unit: mABS) was measured. Here, when the color development intensity is 20 mABS or more, it is determined that BG is colored. Here, the reason why the color is set to 20 mABS or more is that if the color is 20 mABS or more, the coloring can be confirmed visually.
  • mABS color development intensity
  • ⁇ Variation of test line (TL) color development intensity> Regarding the measurement of the TL color development intensity, 100 ⁇ L of the positive sample was dropped onto the sample dropping part of the diagnostic kit in the same manner as described above, and the color development intensity (mABS) of the test line after 5 minutes was measured by an immunochromatographic reader. This measurement was performed a total of 30 times, and the average color development intensity (mABS) was defined as the TL color development intensity.
  • an aqueous ethanol solution containing sodium monochloroacetate 300 g of water, 960 g of ethanol, 122.5 g of sodium monochloroacetate
  • the mixture was stirred at 30 or 50 ° C. for 1 to 12 hours.
  • it was dried to obtain a carboxymethylated sheet-like structure.
  • the sheet-like structure obtained above was adjusted to pH 6.0 to 8.0 with an acetic acid-containing ethanol aqueous solution (acetic acid: 37.5 g, distilled water: 375 g, ethanol: 875 g), and then once with 1375 g of a 70 wt% ethanol aqueous solution.
  • 90 wt% ethanol aqueous solution was washed once with 1250 g, and alcohol was replaced twice with 100 wt% ethanol 1250 g. Then, it is immersed in 1250 g (1 to 100 wt%) of an acetic acid-containing ethanol solution or 1250 g (2 to 5 wt%) of an aqueous ethanol solution, and after stirring for 1 hour, once with 1375 g of a 70 wt% ethanol aqueous solution and once with 1250 g of a 90 wt% ethanol aqueous solution.
  • Example 1 The average substitution degree, catch and release index, basis weight, thickness, and liquid absorption amount of the obtained sheet-like structures (samples 1 to 15) are shown in Table 1 below. These samples 1 to 15 were used as absorption pads.
  • a grass fiber conjugate pad (Ahlstrom, # 8951) was cut into a shape having a height of 10 mm and a length of 300 mm. Then, 1020 ⁇ L of the antibody-sensitized colloidal gold particle dispersion was evenly applied and dried at 50 ° C. for 60 minutes.
  • Sample pad pretreatment Sample pad ((Millipore, C048), PBS buffer containing a large excess of 2.0% by weight BSA (Sigma-Aldrich, A7906) and 2.0% by weight Tween-20®. It was impregnated with (66 mM, pH 7.4), excess liquid was removed, and then dried at 50 ° C. for 60 minutes. Subsequently, it was cut into a shape having a height of 18 mm and a length of 300 mm.
  • BSA Sigma-Aldrich, A7906
  • nitrocellulose membrane coated with capture antibody A nitrocellulose membrane (SHF0900425 manufactured by Millipore) was cut into a shape having a height of 25 mm and a length of 300 mm. Using a liquid coating device (Musashi Engineering Co., Ltd., 300DS), a PBS solution (66 mM, pH 7.4) containing 0.1 wt% anti-hCG- ⁇ mouse antibody (Medix Biochemica Co., Ltd., 6601) was added to 0.1 ⁇ L / mm. It was applied to a portion having a height of 7 mm in proportion.
  • a PBS solution (66 mM, pH 7.4) containing 0.1% by weight of an anti-mouse-rabbit antibody (Z0259 manufactured by Daco) was applied to a portion having a height of 12 mm at a ratio of 0.1 ⁇ L / mm. Subsequently, it was dried at 37 ° C. for 30 minutes.
  • Examples 1 to 15 [Performance evaluation of immunochromatographic diagnostic kit] Samples 1 to 15 shown in Table 1 below were evaluated using each as an absorption pad. In the performance evaluation of the immunochromatographic diagnostic kit using an absorption pad having a width of 5 mm and a thickness of 0.2 mm or more, the developable amount of the developing solution is about 80 to 120 ⁇ L, so 100 ⁇ L was actually developed and evaluated. .. Further, in the case where the kit width is 2.5 mm or the absorption pad having a thickness of less than 0.2 mm is used, the deployable amount of the developing solution is about 30 to 80 ⁇ L, so 50 ⁇ L is actually developed and evaluated. did. The evaluation results are shown in Table 2 below. None of the diagnostic kits varied and did not cause reflux. In addition, all of the diagnostic kits had few detached fibers, and the safety in the process could be ensured.
  • Example 16 was prepared regenerated cellulose continuous filaments sheet-like structure (cupra sheet-like structure) (width 20 cm, 100 g / m 2 basis weight, thickness 0.9 mm).
  • Example 17 Prepare a regenerated cellulose continuous long fiber sheet-like structure (cupra sheet-like structure) (width 20 cm, 100 g / m 2 grain, thickness 1.0 mm), increase the amount of monochloroacetic acid used, and increase the degree of substitution. The treatment was carried out in the same manner as in Reference Example 1 except for the adjustment so as to obtain a sheet-like structure.
  • Example 18 The treatment was carried out in the same manner as in Reference Example 1 except that a regenerated cellulose continuous long fiber sheet-like structure (cupra sheet-like structure) (width 20 cm, 300 g / m 2 basis weight, thickness 4.0 mm) was prepared. A sheet-like structure was obtained.
  • a regenerated cellulose continuous long fiber sheet-like structure (cupra sheet-like structure) (width 20 cm, 300 g / m 2 basis weight, thickness 4.0 mm) was prepared. A sheet-like structure was obtained.
  • Example 19 A sheet-like structure was obtained by performing the same treatment as in Reference Example 1 except that a rayon short fiber sheet-like structure (width 20 cm, 300 g / m 2 basis weight, thickness 12.0 mm) was prepared.
  • Example 20 A sheet-like structure was obtained by performing the same treatment as in Reference Example 1 except that a rayon short fiber sheet-like structure (width 20 cm, 430 g / m 2 basis weight, thickness 6.7 mm) was prepared.
  • Sample 18 had a large variation in test results due to the catch-and-release index being too high. Since the sample 19 was too thick, the flexibility was impaired, the sample 19 could not be firmly bonded, and the variation became large. Since the basis weight of the sample 20 is too large, the flexibility is impaired, the handleability is remarkably lowered, the basis weight cannot be firmly bonded, and the variation becomes large. Backflow has occurred between the glass fiber absorbent pad and the conventionally used cellulose absorbent pad. In addition, when glass fiber is used, there are many desorbed fibers, and it is difficult to ensure safety in the process.
  • the immunochromatographic diagnostic kit using the absorption pad for the immunochromatographic diagnostic kit of the present invention has little variation in color development intensity and is excellent in reproducibility. Furthermore, since backflow can be prevented, it can be suitably used as an absorption pad for various immunochromatographic diagnostic kits.

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Abstract

L'invention concerne un tampon absorbant pour un kit de diagnostic immunochromatographique. Le tampon absorbant démontre une excellente reproductibilité de résultats de test et est conçu pour ne pas provoquer d'écoulement inverse. La présente invention concerne donc un tampon absorbant pour un kit de diagnostic immunochromatographique. Le tampon absorbant comprend des fibres de cellulose carboxyméthylées et est caractérisé en ce que le degré moyen de substitution de groupe hydroxyle dans une unité de glucose constituant les fibres de cellulose carboxyméthylées est de 0,05 à 1,5. La présente invention concerne également un kit de diagnostic immunochromatographique comprenant ledit tampon absorbant.
PCT/JP2020/019144 2019-05-14 2020-05-13 Tampon absorbant pour kit de diagnostic immunochromatographique et kit de diagnostic immunochromatographique WO2020230826A1 (fr)

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CN202080030364.8A CN113767287A (zh) 2019-05-14 2020-05-13 免疫层析诊断试剂盒用吸收垫和免疫层析诊断试剂盒
JP2021519465A JPWO2020230826A1 (ja) 2019-05-14 2020-05-13 イムノクロマト診断キット用吸収パッド及びイムノクロマト診断キット
KR1020217023217A KR20210107781A (ko) 2019-05-14 2020-05-13 이뮤노크로마토 진단 키트용 흡수 패드 및 이뮤노크로마토 진단 키트

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JP2012189346A (ja) 2011-03-09 2012-10-04 Tanaka Kikinzoku Kogyo Kk 吸収パッド
JP2015001398A (ja) * 2013-06-13 2015-01-05 旭化成せんい株式会社 水溶性多糖類を含むイムノクロマト用展開液
JP2017061419A (ja) * 2015-09-24 2017-03-30 国立大学法人 東京大学 カルボキシメチル化されたセルロース繊維及び骨補填材を含有する構造体
US20180217140A1 (en) * 2017-01-27 2018-08-02 Bio-Rad Laboratories, Inc. Lateral flow device
WO2019059812A1 (fr) * 2017-09-21 2019-03-28 Никита Михайлович БЕСПАЛОВ Procédé de plateforme décentralisée pour réaliser et contrôler des campagnes publicitaires à l'aide de contacts intelligents, de technologies de chaîne de blocs et de réseaux neuronaux

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JPS6134615B2 (fr) 1978-09-01 1986-08-08 Yosuke Ookura
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JP2012189346A (ja) 2011-03-09 2012-10-04 Tanaka Kikinzoku Kogyo Kk 吸収パッド
JP2015001398A (ja) * 2013-06-13 2015-01-05 旭化成せんい株式会社 水溶性多糖類を含むイムノクロマト用展開液
JP2017061419A (ja) * 2015-09-24 2017-03-30 国立大学法人 東京大学 カルボキシメチル化されたセルロース繊維及び骨補填材を含有する構造体
US20180217140A1 (en) * 2017-01-27 2018-08-02 Bio-Rad Laboratories, Inc. Lateral flow device
WO2019059812A1 (fr) * 2017-09-21 2019-03-28 Никита Михайлович БЕСПАЛОВ Procédé de plateforme décentralisée pour réaliser et contrôler des campagnes publicitaires à l'aide de contacts intelligents, de technologies de chaîne de blocs et de réseaux neuronaux

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