WO2017009926A1 - Procédé de fixation pour substance à liaison spécifique - Google Patents

Procédé de fixation pour substance à liaison spécifique Download PDF

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Publication number
WO2017009926A1
WO2017009926A1 PCT/JP2015/070026 JP2015070026W WO2017009926A1 WO 2017009926 A1 WO2017009926 A1 WO 2017009926A1 JP 2015070026 W JP2015070026 W JP 2015070026W WO 2017009926 A1 WO2017009926 A1 WO 2017009926A1
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Prior art keywords
specific binding
carrier
binding substance
substance
particles
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PCT/JP2015/070026
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English (en)
Japanese (ja)
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恭一 角田
井上　恵一
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和光純薬工業株式会社
株式会社シノテスト
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Priority to PCT/JP2015/070026 priority Critical patent/WO2017009926A1/fr
Publication of WO2017009926A1 publication Critical patent/WO2017009926A1/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

Definitions

  • the present invention relates to a method for immobilizing a substance that specifically binds to a substance to be measured (hereinafter sometimes abbreviated as a specific binding substance) on the surface of a non-absorbable carrier. More specifically, a specific binding substance and a specific membrane solubilizing agent are mixed in advance, and a mixed solution containing the specific binding substance and the membrane solubilizing agent is applied to the surface of the non-absorbable carrier.
  • Method for immobilizing a chemical binding substance on the surface of a non-absorbable carrier, a non-absorbable carrier obtained by the immobilization method, and a substance to be measured in a sample, characterized by using the non-absorbable carrier It relates to a measurement method.
  • An immunochromatography method (hereinafter sometimes abbreviated as an immunochromatography method) is a diagnosis utilizing the fact that an analyte combined with nanoparticles such as colloidal gold or colored latex develops on a membrane such as nitrocellulose by capillary action. It is a technique. This method is easy to operate, requires a relatively short time of about 10 to 30 minutes, and does not require an expensive device for determination. Therefore, this method is frequently used in clinical settings as an excellent simple diagnostic method. Yes. For example, in the determination of infection with viruses such as influenza virus and RS virus, nasal swabs and throat swabs collected from patients can be used as specimens for a short time and on-site determination. It is popular as a very powerful tool.
  • nanoparticles such as colloidal gold and colored latex are generally used as labeled particles.
  • Labeled particles sensitized with a specific binding substance such as an antibody against the test substance and the test substance are insoluble thin film supports.
  • a specific binding substance such as an antibody against the test substance and the test substance
  • insoluble thin film supports Developed on a membrane such as nitrocellulose by capillary action and sensitized with a specific binding substance by binding to a specific binding substance such as an antibody against a test substance immobilized in advance on an insoluble thin film support A complex of labeled particle-test substance-specific binding substance-immobilized insoluble thin film support is formed, and a positive line is obtained.
  • the immunochromatography method using nanoparticles as described above requires a reaction time of 10 minutes or more in order to determine negative.
  • Patent Document 1 a specific binding substance for a test substance is partially coated on a carrier that does not have water permeability, and after contacting the sample with the surface of the carrier, the sample is removed, By contacting the surface with a specific binding substance that is the same or different from the specific binding substance coated on the surface of the carrier, the particle is brought into contact with the surface, and moved on the carrier in a shorter time than the immunochromatography method. Diagnosis is possible.
  • a specific binding substance for a measurement target substance is applied in advance to a determination unit on a plastic substrate, and a sample containing the measurement target substance is placed on the plastic substrate.
  • the magnetic particles sensitized with a specific binding substance that is the same as or different from the substance to be measured are migrated on the plastic substrate by magnetic force, and in the case of positive, a line is generated in the determination part. Positive or negative result determination is possible with a determination time of 1 minute.
  • the present invention has been made in view of the above situation, and provides a method for immobilizing a specific binding substance capable of suppressing the expression of a non-specific line in an immunotrap method, and a test capable of suppressing the expression of a non-specific line.
  • the object is to provide a device (carrier).
  • the present invention relates to a substance that specifically binds to a substance to be measured (specific binding substance), n-octyl- ⁇ -D-glucoside, n-octyl- ⁇ -D-thioglucoside, n-dodecyl- ⁇ -D.
  • a membrane solubilizer selected from the group consisting of -maltoside, n-nonyl- ⁇ -D-thiomaltoside and n-octanoyl-N-methyl-D-glucamine is mixed in advance, and the specific binding substance and the membrane solubilized It is an invention of a method for immobilizing a specific binding substance, which comprises applying a mixed solution containing an agent to the surface of a non-absorbable carrier and immobilizing a specific binding substance on the surface.
  • the present invention relates to a substance that specifically binds to a substance to be measured (specific binding substance), n-octyl- ⁇ -D-glucoside, n-octyl- ⁇ -D-thioglucoside, n-dodecyl- ⁇ .
  • a membrane solubilizer selected from the group consisting of -D-maltoside, n-nonyl- ⁇ -D-thiomaltoside and n-octanoyl-N-methyl-D-glucamine is mixed in advance, and the specific binding substance and the membrane
  • the present invention relates to a substance that specifically binds to a substance to be measured (specific binding substance), n-octyl- ⁇ -D-glucoside, n-octyl- ⁇ -D-thioglucoside, n-dodecyl- ⁇ .
  • a membrane solubilizer selected from the group consisting of -D-maltoside, n-nonyl- ⁇ -D-thiomaltoside and n-octanoyl-N-methyl-D-glucamine is mixed in advance, and the specific binding substance and the membrane
  • a non-absorbable carrier obtained by applying a mixed solution containing a solubilizer to the surface of a non-absorbable carrier and having a specific binding substance immobilized on the surface is used. It is an invention of a method for measuring a substance to be measured.
  • non-absorbable carrier obtained by using the method for immobilizing a specific binding substance of the present invention for measurement of viruses such as influenza virus and RS virus, non-specific reactions can be suppressed and higher accuracy is achieved. Judgment is possible.
  • the present invention relates to a substance that specifically binds to a substance to be measured (specific binding substance), n-octyl- ⁇ -D-glucoside, n-octyl- ⁇ -D-thioglucoside, n-dodecyl- ⁇ -D.
  • Membrane solubilizer selected from the group consisting of -maltoside, n-nonyl- ⁇ -D-thiomaltoside and n-octanoyl-N-methyl-D-glucamine (hereinafter sometimes abbreviated as membrane solubilizer according to the present invention).
  • a mixture containing the specific binding substance and the membrane solubilizer is applied to the surface of the non-absorbable carrier to immobilize the specific binding substance on the surface. It is an invention of a method for immobilizing a specific binding substance.
  • the present invention also includes a substance that specifically binds to a substance to be measured (specific binding substance) and a membrane solubilizer according to the present invention mixed in advance, and the specific binding substance and the membrane solubilizer It is an invention of a non-absorbable carrier obtained by applying a mixed solution containing a non-absorbable carrier to a surface of the non-absorbable carrier and having a specific binding substance immobilized on the surface.
  • the present invention comprises mixing in advance a substance that specifically binds to the substance to be measured (specific binding substance) and the membrane solubilizer according to the present invention, and the specific binding substance and the membrane solubilizer A non-absorbable carrier having a specific binding substance immobilized on the surface obtained by applying a mixed solution containing a non-absorbable carrier to the surface of the non-absorbable carrier. It is an invention of a measuring method.
  • the substance to be measured according to the present invention may be any substance as long as it is a biological substance such as an organic substance such as protein, carbohydrate, lipid, or nucleic acid, or an inorganic substance.
  • the measurement target substance include, for example, HBs antigen, anti-HBs antibody, HBe antigen, anti-HBe antibody, anti-HBc antibody, anti-HCV antibody, anti-HIV antibody, anti-ATLV antibody, influenza antigen, anti-influenza antibody, RS virus antigen , Anti-RS virus antibodies, adenovirus antigens, anti-adenovirus antibodies, human metapneumovirus antigens, norovirus antigens and other virus-related antigens or antibodies; for example, E.
  • coli O157 antigen anti-Treponema paridum (TP) antibody, anti-cardiolipin antibody, Bacteria-related antigens or antibodies such as mycoplasma antigen, anti-mycoplasma antibody, group A hemolytic streptococcal antigen, anti-streptolysin O antibody (ASO); for example, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immune glob Inflammatory markers such as C-reactive protein (CRP), ⁇ 1-acid glycoprotein, haptoglobin, complement C3, complement C4, rheumatoid factor; for example ⁇ -fetoprotein, CEA, CA19 Tumor markers such as -9; hormones such as human placental chorionic gonadotropin; allergy-related antigens or antibodies such as allergens, allergen-specific IgE antibodies; blood coagulation-related substances such as antithrombin III (ATIII); Fibrinolytic substances
  • the substance that specifically binds to the substance to be measured according to the present invention refers to a substance having affinity for the substance to be measured, for example, protein-protein Examples thereof include those having a property capable of binding to a target substance according to interaction, protein-chemical substance interaction, chemical substance-chemical substance interaction. Specifically, antigen-antibody interaction, sugar chain-lectin interaction, enzyme-inhibitor interaction, protein-peptide chain interaction, chromosome or nucleic acid chain-nucleic acid chain interaction, nucleotide-ligand interaction, receptor- Those that bind based on ligand interactions are included.
  • the specific binding substances immobilized on the carrier may be the same or different as long as they can be bound via the substance to be measured.
  • the membrane solubilizing agent according to the present invention includes n-octyl- ⁇ -D-glucoside, n-octyl- ⁇ -D-thioglucoside, n-dodecyl- ⁇ -D-maltoside, n-nonyl- ⁇ -D-thiomaltoside. And a surfactant selected from n-octanoyl-N-methyl-D-glucamine.
  • These membrane solubilizers are for suppressing non-specific reactions.
  • non-specific reactions can be suppressed by using specific membrane solubilizers.
  • the non-specific reaction can be suppressed only by previously mixing these membrane solubilizers with a specific binding substance and applying this mixed solution to a carrier described later.
  • the non-absorbable carrier according to the present invention is a non-permeable carrier, that is, a solution containing a specific binding substance and a membrane solubilizer or a solution containing a sample (for example, a sample or a sample diluent) is specifically bound. It is desirable that it is non-permeable so that it does not penetrate into the inside of the carrier from the surface on which the substance is coated.
  • the material of the non-absorbable carrier according to the present invention is not limited as long as the specific binding substance can be physically or chemically immobilized and is non-permeable as described above.
  • Examples of the material of such a non-absorbable carrier include polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyacrylate, polymethacrylate, polycarbonate or nylon plastic.
  • the non-absorbable carrier according to the present invention includes, for example, a non-absorbable carrier such as cellulose bonded to the surface of an absorbent carrier (permeable carrier) such as cellulose, or an absorbent. Also included are non-absorbable carriers such as plastic laminated on the surface of a carrier (permeable carrier).
  • non-absorbable carrier As the non-absorbable carrier according to the present invention, a surface on which the specific binding substance is partially coated (supported) by immobilizing the specific binding substance, that is, a part not covered with the specific binding substance. And a non-absorbable carrier having a surface having both the coated portion and the coated portion, or the surface on which the specific binding substance is entirely coated (supported), that is, coated with the specific binding substance.
  • a non-absorbable carrier having a surface with only a portion may be used.
  • a surface on which the specific binding substance is partially coated (supported) that is, a surface having both a part not coated with the specific binding substance and a coated part. It is preferable to use a non-absorbable carrier.
  • the carrier has a surface that is entirely or partially coated with a specific binding substance, and may have any shape and structure as long as a solution (liquid) such as a sample can be brought into contact with the surface.
  • a solution liquid
  • a container, a plate-shaped body, etc. are mentioned.
  • the shape of the recess that is the solution storage portion may be any shape such as a hemispherical shape, a cylindrical shape, a rectangular parallelepiped shape.
  • the inner wall surface of the concave portion may be entirely or partially covered with the specific binding substance, and in particular, it is preferable to partially cover the inner wall surface.
  • the shape of the concave portion is a shape having a flat bottom surface such as a cylindrical shape or a rectangular parallelepiped shape
  • the flat bottom surface may be entirely or partially covered with a specific binding substance, and in particular, partially covered. It is preferable.
  • the container may have a plurality of recesses.
  • the container is preferably a container having at least one recess having a flat bottom surface, and the flat bottom surface is preferably partially covered with a specific binding substance.
  • Examples of the container having at least one recess having a flat bottom include a flat bottom microplate and a tray.
  • the surface shape of the plate-like body may be any shape such as a circle, a rectangle and a square, and the surface is entirely covered with a specific binding substance. Alternatively, it may be partially covered. In addition, it is possible to prevent a solution from flowing down from the surface by forming a channel or a wall on the surface of the plate-shaped body and introducing a solution such as a sample into the channel.
  • the method for mixing the specific binding substance and the membrane solubilizer is not particularly limited as long as it is a method generally used in this field, and for example, phosphate buffered saline (PBS).
  • a buffer solution such as Tris (hydroxymethyl) aminomethane buffer, Good buffer solution, borate buffer solution, etc.
  • a method of mixing a specific concentration of a membrane solubilizer and a specific binding substance into a specific concentration solution Specifically, for example, a method of adding a specific binding substance to a solution in which a membrane solubilizing agent is dissolved in a buffer solution, a membrane solubilizing agent in a solution in which a specific binding substance is dissolved in a buffer solution And a method in which a solution in which a membrane solubilizing agent is dissolved in a buffer and a solution in which a specific binding substance is dissolved in the buffer are mixed.
  • the pH of the buffer solution described above is preferably in the range of 4-12.
  • the concentration of the specific binding substance in the mixed solution containing the specific binding substance and the membrane solubilizing agent after mixing the specific binding substance and the membrane solubilizing agent is usually 0.01 to 10 mg / mL, preferably Is 0.1 to 5 mg / mL.
  • the concentration of the membrane solubilizer in the mixed solution containing the specific binder and membrane solubilizer after mixing the specific binder and membrane solubilizer is usually 0.001-1% (w / vol), preferably 0.001 to 0.1% (w / vol).
  • the immobilization of the specific binding substance on the surface of the non-absorbable carrier can be performed by a physical adsorption method such as hydrophobic bonding or hydrophilic adsorption, or a chemical bonding method using a crosslinking reagent.
  • a physical adsorption method such as hydrophobic bonding or hydrophilic adsorption, or a chemical bonding method using a crosslinking reagent.
  • the immobilization is performed by a physical adsorption method, according to a known method, for example, by bringing a mixed solution containing a specific binding substance and the membrane solubilizing agent according to the present invention into contact with the surface of the non-absorbable carrier. It can be carried out.
  • the non-absorbable carrier is a container
  • the mixture containing the specific binding substance and the membrane solubilizing agent according to the present invention is placed in the recess of the container and allowed to stand, thereby bringing the specific binding substance into contact with the carrier.
  • the adsorption reaction is performed at about 2-40 ° C for about 10 minutes to 1 day
  • the liquid in the recesses is removed by suction and washed with a buffer solution to immobilize the specific binding substance on the non-absorbable carrier. Can be made.
  • non-absorbable carrier By reacting with amino groups, carboxyl groups, thiol groups, aldehyde groups, hydroxyl groups, etc., specific binding substances can be immobilized on non-absorbable carriers.
  • a bivalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, maleimide or the like is added to the recess of the container and allowed to stand and react.
  • a mixture solution containing a specific binding substance and a membrane solubilizing agent according to the present invention is added thereto and allowed to stand to react.
  • the reaction is then stopped by adding a reaction terminator for the crosslinking reaction.
  • the specific binding substance can be immobilized on the non-absorbable carrier by washing with a buffer solution or the like.
  • a method of partially coating the surface of the non-absorbable carrier for example, mixing obtained by previously mixing the specific binding substance and the membrane solubilizer only in the part to be coated with the specific binding substance on the surface.
  • a coating device such as Pulse Injector (registered trademark)
  • coating and fixing by the said method etc. are mentioned.
  • a method of applying a mixed solution obtained by mixing a specific binding substance and a membrane solubilizer in advance or the mixed solution and a crosslinking reagent to a non-absorbable carrier is generally used in this field.
  • the specific binding substance and the membrane solubilizer are previously added using a coating apparatus such as a pulse injector (registered trademark) in the immobilization method of the present invention. It is preferable to apply the mixed solution obtained by mixing or the mixed solution and the crosslinking reagent to the non-absorbable carrier.
  • the coating method using a coating device such as a pulse injector (registered trademark), for example, the method described in Japanese Patent Application Laid-Open No. 2002-176205, that is, a coating device in which a small piezoelectric element is incorporated in a liquid ejection head And a method of applying the liquid by accurately ejecting a highly viscous liquid using the liquid ejection head. More specifically, for example, after filling a liquid ejection head of a coating apparatus with a liquid mixture obtained by previously mixing a specific binding substance and a membrane solubilizer or the liquid mixture and a crosslinking reagent, non-absorption is performed.
  • a coating device such as a pulse injector (registered trademark)
  • the method described in Japanese Patent Application Laid-Open No. 2002-176205 that is, a coating device in which a small piezoelectric element is incorporated in a liquid ejection head
  • a method of applying the liquid by accurately ejecting a highly viscous liquid
  • a liquid ejection head is set above the portion to be coated on the sex carrier, and the mixed liquid or the mixed liquid and the crosslinking reagent are continuously discharged at 3 to 30 pL / droplet.
  • the applied mixed solution or the mixed solution and the crosslinking reagent are naturally dried in a short time, and the specific binding substance is immobilized on the non-absorbable carrier.
  • various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, and nonfat dry milk are brought into contact with a carrier on which a specific binding substance is immobilized.
  • the carrier on which the specific binding substance is immobilized may be blocked by a known method.
  • Specific examples of the blocking solution include phosphate buffered saline (PBS), tris (hydroxymethyl) aminomethane buffer containing 1.0 to 50 mg / mL of protein components such as bovine albumin and casein, Good A buffer solution or the like is used.
  • PBS phosphate buffered saline
  • tris (hydroxymethyl) aminomethane buffer containing 1.0 to 50 mg / mL of protein components such as bovine albumin and casein, Good A buffer solution or the like is used.
  • the pH of the buffer solution described above is preferably in the range of 4-12.
  • Examples of the blocking method include a method in which a non-absorbable carrier on which a specific binding substance is immobilized is immersed in the above-described blocking solution and allowed to stand at 1 to 40 ° C. for 10 minutes to 72 hours. After blocking, drying under reduced pressure may be performed for stabilization. Examples of the drying method under reduced pressure include a method of standing for 1 to 72 hours under a reduced pressure of 0.1 to 10 Pa.
  • the non-absorbable carrier obtained by the immobilization method of the present invention can be used, for example, as a carrier in the above-described immunotrap method. More specifically, after the sample is brought into contact with the surface of the non-absorbable carrier, the specific binding substance for the substance to be measured is the same as the specific binding substance immobilized on the non-absorbable carrier or Contact the particles with different specific binding substances immobilized, move the carrier so that the particles move along the surface, and determine the presence or absence of the measurement target substance from the distribution state of the particles on the surface It can be used as a carrier for the method.
  • particles there are particles generally used for indirect agglutination reaction.
  • organic polymer particles such as liposome, latex particles, gelatin particles, polyacrylamide particles, microcapsules, emulsions, inorganic polymer particles such as glass beads, silica beads, bentonite, other artificial particles, erythrocytes, etc.
  • inorganic polymer particles such as glass beads, silica beads, bentonite, other artificial particles, erythrocytes, etc.
  • magnetic particles can be used as the particles.
  • the magnetic particles may be particles that are magnetized at least while a magnet is applied from the outside.
  • the magnetic particles include ferromagnetic metals such as iron, cobalt and nickel, alloys containing these ferromagnetic metals, particles containing a ferromagnetic metal or an alloy containing a ferromagnetic metal in a non-magnetic material, and ferromagnetic metals.
  • Particles coated with a high molecular weight material particles coated with a ferromagnetic material with particles of a high molecular weight material such as polystyrene, silica gel, gelatin, polyacrylamide, etc., and closed bag-shaped materials such as erythrocytes, liposomes or microcapsules Examples include particles encapsulating a ferromagnetic material.
  • the magnetic particles have a property of being magnetized while a magnet is applied from the outside and demagnetizing quickly by blocking the magnet from the outside.
  • particles colored by coating a dye or dispersing or encapsulating the dye in the particles may be used, and the dye may be a fluorescent dye.
  • the particle diameter of the particles is usually 0.001 to 1000 ⁇ m, preferably 0.01 to 100 ⁇ m, more preferably 0.5 to 10 ⁇ m.
  • the specific gravity of the particles may be any specific gravity that settles in the dispersion medium. For example, a specific gravity of 1 to 10 is preferable.
  • particles having a specific binding substance immobilized thereon are used.
  • the specific binding substance is described above. It can be carried out on the surface of the particles by a physical adsorption method such as hydrophobic bonding or hydrophilic adsorption, a chemical bonding method such as covalent bonding, or a combination of these methods.
  • the specific binding substance and the particles may be mixed and brought into contact with each other in a solution such as a buffer solution according to a known method. It can.
  • a specific binding substance and particles are brought into contact with each other by mixing and stirring in a solution such as a buffer solution, and an adsorption reaction is performed at about 2 to 40 ° C. for about 10 minutes to 1 day, and then the obtained particles are buffered.
  • the specific binding substance can be immobilized on the particles by washing with a liquid or the like.
  • the specific binding substance can be immobilized on the particles.
  • a bivalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, maleimide or the like is added to a buffer solution containing particles, and the mixture is stirred and reacted. Subsequently, a specific binding substance is added to this, and it is made to react by stirring. In some cases, the reaction is then stopped by removing the crosslinking reagent by treatment such as dialysis or gel filtration or adding a reaction stopper for the crosslinking reaction.
  • the specific binding substance can be immobilized on the particles by washing the obtained particles with a buffer solution or the like.
  • the sensitivity can be easily changed according to the concentration of the substance to be measured in the sample. For example, when a specific binding substance is immobilized on the particles, if a high concentration of the specific binding substance is used, the amount of immobilization increases and the sensitivity can be increased.
  • various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, skim milk powder, etc. are brought into contact with particles on which a specific binding substance has been immobilized in order to suppress nonspecific reactions.
  • the particles may be masked by a known method such as For masking, for example, particles having a specific binding substance immobilized thereon are added to a buffer solution containing various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, and left to stand. It can carry out by coating with etc.
  • the above-mentioned sample refers to a liquid sample in which the above-mentioned measurement target substance may be present and the presence / absence of the measurement target substance may be confirmed or quantified.
  • Body fluids such as human or animal blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites, amniotic fluid, etc .
  • organs such as human or animal brain, hair, skin, nails, muscles, or Extracts such as neural tissues; human or animal fecal extracts or suspensions; cells or fungal extracts; plant extracts and the like.
  • a specific binding substance is immobilized, and the surface partially covered with the specific binding substance, that is, the specific binding substance is covered.
  • An example is a method in which a sample suspected of the presence of a substance to be measured is brought into contact with the surface of a carrier having a surface having both an uncoated part and a coated part (hereinafter abbreviated as the first measuring method of the present invention). May be.)
  • the sample can be brought into contact with the inner wall surface of the container by adding the sample to the recess of the container.
  • the non-absorbable carrier is, for example, a plate-like body as described above, the sample may be contacted by dropping a sample on the surface of the plate-like body.
  • the sample can be diluted with, for example, a diluent and brought into contact with a non-absorbable carrier.
  • a diluted solution of the sample various buffers such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, phosphate buffered saline, or physiological saline can be used.
  • the pH of the buffer solution described above is preferably in the range of 4-12.
  • Sample dilutions include bovine serum albumin, human serum albumin, various proteins such as casein or salts thereof, various salts such as sodium chloride, various sugars, various animal sera such as skim milk powder, normal rabbit serum, and azide.
  • Various preservatives such as sodium, nonionic surfactants, cationic surfactants, anionic surfactants, various surfactants such as amphoteric surfactants, n-octyl- ⁇ -D-glucoside, etc.
  • Additives such as membrane solubilizers can be appropriately added and used.
  • the concentration when these additives are added is not particularly limited, but is usually 0.001 to 10% (w / vol), preferably 0.01 to 5% (w / vol).
  • surfactant examples include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene.
  • Nonionic surfactants such as phytosterol, polyoxyethylene phytostanol, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil, polyoxyethylene lanolin, such as polyoxyethylene alkyl ether acetate, polyoxyethylene
  • anionic surfactants such as alkyl ether sulfates, and amphoteric surfactants such as betaine acetate.
  • membrane solubilizers include n-octyl- ⁇ -D-glucoside, n-octyl- ⁇ -D-thioglucoside, n-dodecyl- ⁇ -D-maltoside, and n-nonyl- ⁇ -D-thiomaltoside. N-octanoyl-N-methyl-D-glucamine and the like.
  • the sample brought into contact with this surface is removed.
  • the removal of the sample that has been brought into contact with the surface of the non-absorbent carrier that is coated with the specific binding substance allows the sample absorber made of a water-absorbing material to come into contact with the sample on the surface of the non-absorbent carrier to absorb the sample. Or by sucking the sample on the surface of the non-absorbable carrier with a pipette or the like, or turning the non-absorbable carrier over and dropping the sample down.
  • the specific binding substance is used so that the sample moves on the portion coated with the specific binding substance. It is preferable that the sample is absorbed or sucked from the horizontal direction of the portion covered with. Thereby, a measurement object substance can be measured with higher sensitivity.
  • Sample absorbents made of water-absorbing materials include, for example, papers such as filter paper, paper towels, tissue papers, sintered bodies made of, for example, polyethylene or polystyrene, sponges made of, for example, polyvinyl alcohol or polyurethane, for example, rayon, polyester, etc.
  • the sample When the sample is removed in this manner, when the sample is a blood sample (whole blood sample), red blood cells in the blood sample are removed from the surface of the non-absorbable carrier on the surface of the non-absorbable carrier. In contrast, the substance to be measured in the blood sample remains bound to the specific binding substance immobilized on the surface of the non-absorbable carrier. And since the particle
  • the surface of the carrier may be washed with the above-described sample dilution or buffer solution.
  • a specific binding substance that is the same or different from the specific binding substance immobilized on the non-absorbable carrier was immobilized on the specific substance to be measured.
  • the particles are brought into contact with the surface of the non-absorbable carrier from which the sample has been removed.
  • the particles on which the specific binding substance is immobilized particles on which the measurement target substance or its analog is immobilized may be used.
  • the analog of the measurement target substance is a part of the measurement target substance, a combination of another substance with the measurement target substance, a part of the structure of the measurement target substance substituted, and the like. It is a substance that has a structure of a portion that binds to the specific binding substance and can bind to the specific binding substance.
  • the particles can be dispersed, for example, in a suitable dispersion medium and brought into contact with a non-absorbable carrier.
  • a suitable dispersion medium various buffer solutions such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, phosphate buffered saline, or physiological saline can be used.
  • the pH of the buffer solution described above is preferably in the range of 4-12.
  • the additives described in the section of the sample diluent described above can be appropriately added to the particle dispersion medium.
  • concentration at which the additive is added is not particularly limited, but is preferably 0.001 to 10% (w / vol), and more preferably 0.01 to 5% (w / vol).
  • FIG. 1 is a view showing a flat bottom microplate 1 used as a carrier, in which a half half 3 of a flat bottom 2 of each well is coated with a specific binding substance.
  • 1A is a perspective view of the flat bottom microplate 1 (the hatched portion 3 is a portion covered with the specific binding substance)
  • FIG. 1B is a plan view of the flat bottom microplate 1. (The hatched portion 3 is a portion covered with the specific binding substance).
  • C of FIG. 1 is a view of the well as seen from above when the flat bottom microplate 1 is used and the measurement is performed on the sample in which the measurement target substance is present (“positive” sample).
  • FIG. 1 is a view showing a flat bottom microplate 1 used as a carrier, in which a half half 3 of a flat bottom 2 of each well is coated with a specific binding substance.
  • 1A is a perspective view of the flat bottom microplate 1 (the hatched portion 3 is a portion covered with the specific binding substance)
  • FIG. 1B is
  • 1D is a view of a well as seen from above when a flat bottom microplate 1 is used and measurement is performed on a sample in which a measurement target substance does not exist (“negative” sample).
  • negative sample a sample in which a measurement target substance does not exist
  • the particles 4 that have migrated from the portion of the flat bottom surface 2 of the well that is not coated with the specific binding substance are trapped in the specific binding substance coating portion.
  • the arrow in a figure shows the moving direction of the particle
  • FIG. 2 shows a rectangular parallelepiped transparent container 5 having a bottom surface and a bottom surface having a portion 6 (hereinafter sometimes abbreviated as a belt-shaped covering portion) covered in a band shape with a specific binding substance.
  • FIG. 2A is a perspective view of the container 5 (the hatched portion 6 is a portion covered with the specific binding substance), and
  • FIG. 2B is a plan view of the container 5. (The hatched portion 6 is a portion covered with the specific binding substance).
  • FIG. 2C is a view of the container 5 as viewed from above when a rectangular parallelepiped transparent container 5 having a rectangular bottom surface is used and a sample containing a measurement target substance (a “positive” sample) is measured. It is.
  • FIG. 1 is a perspective view of the container 5 (the hatched portion 6 is a portion covered with the specific binding substance)
  • FIG. 2B is a plan view of the container 5. (The hatched portion 6 is a portion covered with the specific binding substance).
  • 2D is a view of the container 5 as viewed from above when a rectangular parallelepiped transparent container 5 having a rectangular bottom surface is used and a sample (“negative” sample) in which the measurement target substance does not exist is measured. It is.
  • the particles 4 that have moved from the portion not covered with the specific binding substance on the bottom surface are trapped in the band-shaped covering portion 6.
  • the arrow in a figure shows the moving direction of the particle
  • FIG. 3 is a diagram showing a plate-like body 7 having a rectangular surface shape and having a surface having strip-shaped covering portions 8 and 8 ′ made of two different types of specific binding substances.
  • 3A is a perspective view of the plate-like body 7 (hatched portions 8 and 8 ′ are portions coated with different specific binding substances, respectively), and FIG. 3B shows the plate.
  • FIG. 2 is a plan view of the shape (hatched portions 8, 8 ′ are portions coated with different specific binding substances, respectively).
  • FIG. 3C shows a plate in the case where measurement is performed on a sample in which a rectangular plate-like body 7 is used and two types of measurement target substances exist (samples each having a “positive” measurement target substance). It is the figure which looked at 7 from the upper part.
  • FIG. 3 shows a plate in the case where measurement is performed on a sample in which a rectangular plate-like body 7 is used and two types of measurement target substances exist (samples each having a “positive” measurement target substance). It is the figure
  • 3D is a view of the plate-like body 7 as viewed from above when the rectangular plate-like body 7 is used and the measurement is performed on a sample in which the measurement target substance does not exist (“negative” sample). .
  • the particles 4 that have moved from the portion not covered with the specific binding substance on the surface are trapped in the band-like covering portions 8 and 8 ′ corresponding to the respective substances to be measured.
  • the arrow in a figure shows the moving direction of the particle
  • FIG. 4 shows a plate-like body 9 having a rectangular surface shape, a flow path (groove) 10 having a rectangular cross-sectional shape provided on the surface, and two different types on the bottom surface of the flow path (groove) 10. It is the figure which showed what formed the strip
  • FIG. 4C shows a sample in which a rectangular plate-like body 9 having a flow path (groove) 10 on the surface is used, and two types of measurement target substances are present (each measurement target substance is a “positive” sample). It is the figure which looked at this plate-shaped object 9 at the time of measuring about ().
  • FIG. 4D shows a case where a rectangular plate-like body 9 having a flow path (groove) 10 provided on the surface thereof is used and a sample (“negative” sample) in which no measurement target substance exists is measured. It is the figure which looked at the plate-shaped object 9 from upper direction.
  • the particles 4 that have moved from the portion not covered with the specific binding substance on the bottom surface of the channel (groove) 10 are the strip-like covering portions 11, 11 corresponding to the respective substances to be measured. Trapped by '.
  • the arrow in a figure shows the moving direction of the particle
  • the surface of the non-absorbable carrier that is partially coated with the specific binding substance is coated from a portion that is not covered with the specific binding substance.
  • Move the particles to the part Examples of a method for moving particles from a portion not coated with a specific binding substance to a portion coated with a specific binding substance include a method of acting a magnet and a method of tilting a non-absorbable carrier.
  • the above-described magnetic particles are used.
  • the magnet is caused to move so that the magnetic particles move from a portion not covered with the specific binding substance to a covered portion on the surface partially covered with the specific binding substance.
  • a magnet is allowed to act before or while the magnetic particles are brought into contact with the surface of the non-absorbable carrier. May be.
  • the magnet When the surface of the non-absorbable carrier is partially coated with a specific binding substance, the magnet may be disposed anywhere as long as the magnetic particles can be moved in the desired direction, for example, What is necessary is just to arrange
  • any magnet may be used as long as it generates a magnetic field and magnetizes the magnetic particles, and a permanent magnet, an electromagnet, or the like may be used.
  • the magnetic flux density is usually 5 to 100 gauss, although it depends on the interaction between the magnetic particles used and the carrier surface.
  • the substance to be measured binds to the specific binding substance immobilized on the non-absorbable carrier.
  • the magnetic particles are attracted by the magnet and move in the direction of the magnet along the surface of the non-absorbent carrier.
  • specific binding immobilized on the surface is performed.
  • the movement of the magnetic particles depends on the interaction between the particles and the carrier surface and the strength of the magnetic field, and the magnetic particles move rapidly in the direction of the magnet. To do.
  • the magnetic particles are divided depending on whether the measurement target substance is bound to the specific binding substance immobilized on the surface or not. There is a big difference in the moving speed.
  • the specific binding substance immobilized on the surface does not bind the measurement target substance, so that the magnetic particles do not show affinity and the specific binding substance is fixed. It moves quickly in the direction of the magnet in the same manner as in the unstructured area. Therefore, when the measurement target substance is not present in the sample, the magnetic particles gather at a position close to the magnet, that is, at the end of the surface of the non-absorbable carrier.
  • the specific binding substance immobilized on the surface binds the measurement target substance, so that the magnetic particles are not passed through the measurement target substance and the specific binding substance. Binding to the absorbent carrier stops or slows down significantly. Therefore, when the substance to be measured exists in the sample, the magnetic particles collect on the surface of the surface covered with the specific binding substance.
  • the magnetic particles added to the container settle down below the recess of the container due to its specific gravity. At this time, the sedimentation of the magnetic particles may be promoted by applying a magnet from the top of the container to the bottom.
  • the particles When moving by tilting the non-absorbable carrier, the particles are coated from the part of the non-absorbent carrier that is partially coated with the specific binding substance and not covered by the specific binding substance.
  • the non-absorbent carrier is tilted so as to move to the part where the particles are, and the particles are moved by gravity.
  • the angle at which the non-absorbable carrier is tilted is covered from the portion of the surface where the particles are partially coated with the specific binding substance of the non-absorbing carrier by gravity, which is not covered with the specific binding substance.
  • the angle may be an angle that moves to the portion, and an angle of 90 ° or less may be selected as appropriate. An angle between 25 ° and 90 ° is preferable, and an angle between 45 ° and 65 ° is particularly preferable. preferable.
  • the carrier may be tilted before or while the particles are brought into contact with the carrier surface.
  • the non-absorbable carrier When moving the particles by gravity, as described above, for example, the non-absorbable carrier may be arranged with the direction to be moved down, and more specifically, for example, a half of the surface is bound to the specific binding substance.
  • the particles are moved so that the particles move from the portion not coated with the specific binding substance on the surface to the coated portion (the arrow direction in FIG. 1). It is preferred to tilt the absorbent carrier.
  • the substance to be measured binds to the specific binding substance immobilized on the non-absorbable carrier.
  • the non-absorbing carrier is tilted so that the particles move along the surface of the non-absorbing carrier coated with the specific binding substance, the particles are tilted along the surface of the non-absorbing carrier by gravity.
  • the measurement target substance bound to the specific binding substance immobilized on the surface is encountered in the process, the particle passes through the measurement target substance and the specific binding substance bound thereto. It will bind to the carrier and stop moving, or the movement will be significantly slowed down.
  • the movement of the particle depends on the interaction between the particle and the carrier surface and the angle at which the carrier is inclined, and the particle moves quickly downward in the inclination.
  • the particle binding of the specific binding substance immobilized on the surface depends on whether the target substance is bound or not. A big difference is made in the moving speed.
  • the specific binding substance immobilized on the surface does not bind the measurement target substance, so the particles do not show affinity and the specific binding substance is immobilized. As with the non-applied area, it immediately moves downward in the inclination. Therefore, when the measurement target substance does not exist in the sample, the particles gather at the lower end of the carrier.
  • the specific binding substance immobilized on the surface binds the measurement target substance, so that the particles are not absorbed through the measurement target substance and the specific binding substance. Binds to the sex carrier and stops or slows down significantly. Therefore, when the measurement target substance is present in the sample, the particles collect on a portion of the surface covered with the specific binding substance.
  • the non-absorbable carrier When the non-absorbable carrier is tilted to move the particles from the portion not coated with the specific binding substance of the non-absorbable carrier to the portion coated with the non-absorbable carrier, The interaction is weak, and the moving speed depends almost on the angle of tilting the non-absorbing carrier. Therefore, when a high moving speed is required, that is, when a measurement result is obtained in a short time, the angle at which the non-absorbent carrier is tilted may be increased. Moreover, you may change the angle which inclines a nonabsorbable support
  • the specific binding substance when a specific binding substance is immobilized on the surface of a non-absorbable carrier, the specific binding substance is applied and immobilized so that the surface is partially coated with the specific binding substance. It is preferable to do. This is because, in the case where the measurement target substance is present in the sample, the determination of being “positive” is made by comparing the image of the part coated with the specific binding substance with the image of the part not covered. It becomes clearer and it becomes easier to discriminate between “positive” and “negative”, that is, the presence or absence of the substance to be measured in the sample.
  • “partial” means that the specific binding substance is unevenly distributed and is not entirely covered with the specific binding substance over the entire surface.
  • the shape and area of the portion coated with the specific binding substance are not particularly limited as long as the presence or absence of the measurement target substance in the sample can be determined.
  • Examples of the partial coating include an example in which one half of the carrier surface is coated, or the surface of the non-absorbent carrier is coated in various shapes such as strips, letters, and figures.
  • each surface of the non-absorbable carrier is measured.
  • a coating portion with a specific binding substance for the target substance can be provided.
  • the presence or absence of the substance to be measured is determined from the distribution state of the particles on the surface.
  • the particle distribution state on the surface of the non-absorbable carrier in other words, the particle distribution image can be confirmed by the naked eye or by an optical reading device such as a microplate reader based on absorbance measurement or pattern recognition.
  • a specific binding substance is immobilized and the surface partially covered with the specific binding substance, that is, a specific binding substance is used.
  • the surface of the non-absorbable carrier is a mixture of particles and a sample in which a specific binding substance is immobilized on the surface of a non-absorbable carrier having a surface that has both a portion that is not coated with a binding substance and a portion that is coated. (Hereinafter, it may be abbreviated as the second measurement method of the present invention.).
  • the mixture After contacting the surface of the non-absorbable carrier with a mixture of particles and sample in which the specific binding substance is immobilized, the mixture is moved over the part of the carrier surface coated with the specific binding substance. The presence / absence of the substance to be measured is determined from the distribution state of the particles on the surface of the carrier.
  • the particles bind to the measurement target substance in the sample.
  • the particles When brought into contact with the surface of the non-absorbable carrier, the particles bind to the specific binding substance immobilized on the surface of the carrier via the substance to be measured.
  • the particles are first bound to the surface of the non-absorbable carrier via the substance to be measured. The movement is stopped or remarkably slowed by the trapped particles, and the particles collect on the part of the support surface coated with the specific binding substance.
  • the particle does not bind the measurement target substance, so when the mixture of the particle and the sample is brought into contact with the surface of the non-absorbable carrier, The particles do not bind to the specific binding substance that has been immobilized on the surface of the carrier. Therefore, even if the mixture of particles and sample is removed from the carrier surface, and the particles are moved along the surface in contact with the carrier surface, the particles will pass over the carrier surface and quickly move in the direction or inclination of the magnet. It moves downward and gathers at a position close to the magnet, that is, at the end of the surface of the carrier, or at the lower carrier of the carrier. Therefore, according to such a measuring method, the same result as the distribution state of the “positive” or “negative” particles obtained by the first measuring method can be obtained.
  • the measuring method of the present invention can be used for a confirmation test.
  • the confirmation test is a method for confirming whether a “positive” image is obtained by measurement, whether the image is really due to the presence of a substance to be measured or due to a non-specific agglutination reaction.
  • FIG. 5 shows two polystyrene plates 14 and 14 ′ (width 2 mm, depth 33 mm, thickness 0.5 mm) bonded to both ends on a polystyrene substrate 16 (width 10 mm, depth 33 mm, thickness 1 mm).
  • a plate-like body 13 having a flow path (groove) 10 (width 6 mm, depth 33 mm, height 0.5 mm) on the surface was prepared.
  • an antibody solution was prepared by diluting the antibody so that the antibody concentration was 1 mg / mL.
  • a pulse injector coating device manufactured by Cluster Technology Co., Ltd.
  • the antibody solution is formed in a line of about 1 mm at a position 14 mm from the front of the bottom surface of the flow path (groove) 10 of the polystyrene plate 13. Applied.
  • the polystyrene plate-like body 13 coated with the antibody is applied to a blocking solution (50 mM Tris-HCl buffer (TBS; pH 7.0), 0.5% casein, 5% lactose, 0.09% sodium azide). It was immersed at 6 ° C. overnight and subjected to blocking treatment. Next, after removing water from the polystyrene plate 13 subjected to the blocking treatment, it was left to stand overnight under reduced pressure and dried. The dried polystyrene plate 13 was attached with an absorbent body made of polyolefin fibers on one side and a cover 12 having a dropping port on the other side to obtain a test device.
  • a blocking solution 50 mM Tris-HCl buffer (TBS; pH 7.0), 0.5% casein, 5% lactose, 0.09% sodium azide.
  • influenza B monoclonal antibody 4.5 mg / mL was added.
  • the resulting solution was sensitized at 37 ° C. for 2 hours while rotating and shaking, followed by blocking solution (50 mM Tris-HCl buffer (TBS; pH 8.0), 0.5% casein, 0% 0.09% sodium azide) was added and subjected to blocking treatment at 37 ° C. for 1 hour while rotating and shaking.
  • blocking solution 50 mM Tris-HCl buffer (TBS; pH 8.0), 0.5% casein, 0% 0.09% sodium azide
  • a magnet was pressed against the magnetic particles after the blocking treatment to collect the magnetic particles, and then the supernatant of the blocking solution was sucked with an aspirator.
  • Influenza virus measurement method Influenza A virus culture solution (4.0 ⁇ 10 7 pfu / mL) or influenza B virus culture solution (2.2 ⁇ 10 7 pfu / mL) The sample was diluted with 50 mM Tris-HCl buffer (TBS; pH 8.0) containing casein to prepare samples of 40-fold dilution, 80-fold dilution, 160-fold dilution, 320-fold dilution, and 640-fold dilution.
  • TSS Tris-HCl buffer
  • Example 1 the test device of Example 1 was set on a slide plate of PULLUNO (manufactured by Wako Pure Chemical Industries, Ltd.), and the above five types of samples were diluted with a specimen diluent (50 mM Tris-HCl buffer (pH 8) containing a surfactant). 0.0), 0.5 M NaCl, 0.5% casein, 0.0015% brilliant blue, 0.09% sodium azide) 60 ⁇ L of diluted sample 10 times each, After visually confirming that the diluted liquid flowed on the measurement lane and was completely absorbed by the absorber, 50 ⁇ L of the above magnetic particle liquid was dropped into the dropping port. The measurement was started by pushing the PULLUNO slide plate fully to the magnet side.
  • a specimen diluent 50 mM Tris-HCl buffer (pH 8) containing a surfactant.
  • 0.0 0.5 M NaCl, 0.5% casein, 0.0015% brilliant blue, 0.09% sodium azide
  • FIG. 5 shows two polystyrene plates 14 and 14 ′ (width 2 mm, depth 33 mm, thickness 0.5 mm) bonded to both ends on a polystyrene substrate 16 (width 10 mm, depth 33 mm, thickness 1 mm).
  • a plate-like body 13 having a flow path (groove) 10 (width 6 mm, depth 33 mm, height 0.5 mm) on the surface was prepared.
  • the anti-influenza A monoclonal antibody or anti-influenza B monoclonal antibody was diluted so that the antibody concentration of the antibody was 1 mg / mL. It was set as the solution.
  • the antibody solution is formed in a line of about 1 mm at a position 14 mm from the front of the bottom surface of the flow path (groove) 10 of the polystyrene plate 13. Applied.
  • the polystyrene plate-like body 13 coated with the antibody was mixed with a blocking solution (50 mM Tris-HCl buffer (TBS; pH 7.0), 0.5% casein, 5% lactose, 0.09% sodium azide). It was immersed in a mixed solution of 0.03% n-octyl- ⁇ -D-thioglucoside aqueous solution at 6 ° C. overnight to perform a blocking treatment. Next, after removing water from the polystyrene plate 13 subjected to the blocking treatment, it was left to stand overnight under reduced pressure and dried. The dried polystyrene plate 13 was attached with an absorbent body made of polyolefin fibers on one side and a cover 12 having a dropping port on the other side to obtain a test device.
  • a blocking solution 50 mM Tris-HCl buffer (TBS; pH 7.0), 0.5% casein, 5% lactose, 0.09% sodium azide. It was immersed in a mixed solution of 0.03%
  • Experimental Example 2 Measurement of Influenza Virus Influenza virus was measured in the same manner as in Experimental Example 1 except that the test device of Comparative Example 1 was used. The determination results are shown in Tables 1 and 2.
  • FIG. 5 shows two polystyrene plates 14 and 14 ′ (width 2 mm, depth 33 mm, thickness 0.5 mm) bonded to both ends on a polystyrene substrate 16 (width 10 mm, depth 33 mm, thickness 1 mm).
  • a plate-like body 13 having a flow path (groove) 10 (width 6 mm, depth 33 mm, height 0.5 mm) on the surface was prepared.
  • the anti-influenza A monoclonal antibody or anti-influenza B monoclonal antibody was diluted so that the antibody concentration of the antibody was 1 mg / mL. It was set as the solution.
  • the polystyrene plate 13 was immersed in a 0.03% aqueous solution of n-octyl- ⁇ -D-thioglucoside at 25 ° C. for 2 hours, and the surface of the polystyrene plate was n-octyl- ⁇ -D-. Coated with thioglucoside.
  • the antibody solution is lined approximately 1 mm at a position 14 mm from the front of the bottom surface of the flow path (groove) 10 of the polystyrene plate 13. It was applied to the shape.
  • the polystyrene plate-like body 13 coated with the antibody is applied to a blocking solution (50 mM Tris-HCl buffer (TBS; pH 7.0), 0.5% casein, 5% lactose, 0.09% sodium azide). It was immersed at 6 ° C.
  • the dried polystyrene plate 13 was attached with an absorbent body made of polyolefin fibers on one side and a cover 12 having a dropping port on the other side to obtain a test device.
  • Experimental Example 3 Measurement of Influenza Virus Influenza virus was measured in the same manner as in Experimental Example 1, except that the test device of Comparative Example 2 was used. The determination results are shown in Tables 1 and 2.
  • FIG. 5 shows two polystyrene plates 14 and 14 ′ (width 2 mm, depth 33 mm, thickness 0.5 mm) bonded to both ends on a polystyrene substrate 16 (width 10 mm, depth 33 mm, thickness 1 mm).
  • a plate-like body 13 having a flow path (groove) 10 (width 6 mm, depth 33 mm, height 0.5 mm) on the surface was prepared.
  • the anti-influenza A monoclonal antibody or anti-influenza B monoclonal antibody was diluted so that the antibody concentration of the antibody was 1 mg / mL. It was set as the solution.
  • the antibody solution was applied in a line shape at a position 14 mm from the front of the bottom surface of the flow path (groove) 10 of the polystyrene plate-like body 13 using a pulse injector application device (manufactured by Cluster Technology Co., Ltd.).
  • the polystyrene plate 13 coated with the antibody was immersed in a 0.03% aqueous solution of n-octyl- ⁇ -D-thioglucoside at 25 ° C. for 2 hours, and the surface of the polystyrene plate 13 was n-octyl- ⁇ -. D-thioglucoside was coated. Subsequently, after removing the water
  • a blocking solution 50 mM Tris-HCl buffer (TBS; pH 7.0
  • Experimental Example 4 Measurement of Influenza Virus Influenza virus was measured in the same manner as in Experimental Example 1, except that the test device of Comparative Example 3 was used. The determination results are shown in Tables 1 and 2.
  • the anti-influenza A monoclonal antibody was diluted so that the antibody concentration of the antibody was 0.2 mg / mL to prepare an antibody solution.
  • the antibody solution is formed in a line of about 1 mm at a position 14 mm from the front of the bottom surface of the flow path (groove) 10 of the polystyrene plate 13. Applied.
  • the polystyrene plate-like body 13 coated with the antibody is applied to a blocking solution (50 mM Tris-HCl buffer (TBS; pH 7.0), 0.5% casein, 5% lactose, 0.09% sodium azide). It was immersed at 6 ° C. overnight and subjected to blocking treatment. Next, after removing water from the polystyrene plate 13 subjected to the blocking treatment, it was left to stand overnight under reduced pressure and dried. The dried polystyrene plate 13 was attached with an absorbent body made of polyolefin fibers on one side and a cover 12 having a dropping port on the other side to obtain a test device.
  • a blocking solution 50 mM Tris-HCl buffer (TBS; pH 7.0), 0.5% casein, 5% lactose, 0.09% sodium azide.
  • Experimental Examples 5 to 12 Measurement of Influenza Virus Influenza virus was measured in the same manner as in Experimental Example 1 except that the test devices of Examples 2 to 6 and Comparative Examples 4 to 6 were used. Table 3 shows the results of the determination.
  • the anti-influenza A monoclonal antibody was diluted so that the antibody concentration of the antibody was 0.2 mg / mL to prepare an antibody solution.
  • the antibody solution is formed in a line of about 1 mm at a position 14 mm from the front of the bottom surface of the flow path (groove) 10 of the polystyrene plate 13.
  • the polystyrene plate-like body 13 coated with the antibody is applied to a blocking solution (50 mM Tris-HCl buffer (TBS; pH 7.0), 0.5% casein, 5% lactose, 0.09% sodium azide). It was immersed at 6 ° C.
  • the dried polystyrene plate 13 was attached with an absorbent body made of polyolefin fibers on one side and a cover 12 having a dropping port on the other side to obtain a test device.
  • a non-absorbable carrier obtained by the immobilization method of the present invention is used to measure, for example, influenza virus, a non-specific reaction can be suppressed, and the measurement target substance can be accurately obtained. It turns out that it can be detected.
  • a non-absorbable carrier obtained by using the method for immobilizing a specific binding substance of the present invention for example, for measurement of influenza virus or the like, a non-specific reaction can be suppressed, and “positive” or It becomes possible to determine “negative”.

Abstract

La présente invention vise à concevoir un procédé de fixation pour une substance à liaison spécifique, capable de supprimer un phénomène dans lequel une ligne positive apparaît même dans une mesure à blanc dans un procédé immuno-TRAP, et à concevoir un dispositif de test (support) capable de supprimer l'apparition d'une ligne positive. La présente invention concerne : un procédé de fixation pour une substance à liaison spécifique, caractérisé par les étapes consistant à prémélanger une substance (substance à liaison spécifique) qui se lie de manière spécifique à une substance à mesurer et un agent de solubilisation de membrane sélectionné dans le groupe constitué du n-octyl-β-D-glucoside, du n-octyl-β-D-thioglucoside, du n-dodecyl-β-D-maltoside, du n-nonyl-β-D-thiomaltoside et de la n-octanoyl-N-méthyl-D-glucamine, et à appliquer, sur une surface d'un support non absorbant, un mélange liquide contenant la substance à liaison spécifique et l'agent de solubilisation de membrane, de façon à fixer ainsi la substance à liaison spécifique à la surface; un support non absorbant obtenu à l'aide du procédé de fixation; et un procédé de mesure pour une substance à mesurer dans un échantillon, caractérisé par l'utilisation du support non absorbant.
PCT/JP2015/070026 2015-07-13 2015-07-13 Procédé de fixation pour substance à liaison spécifique WO2017009926A1 (fr)

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JP2019066472A (ja) * 2017-09-29 2019-04-25 三洋化成工業株式会社 免疫測定用試薬、免疫測定用キット及び免疫測定方法
JP7211748B2 (ja) 2017-09-29 2023-01-24 三洋化成工業株式会社 免疫測定用試薬、免疫測定用キット及び免疫測定方法
CN111886498A (zh) * 2018-03-16 2020-11-03 富士胶片株式会社 试剂盒、测定试剂盒及测定方法
CN112166315A (zh) * 2018-12-17 2021-01-01 松下知识产权经营株式会社 修饰粒子、修饰粒子的制造方法及检测装置

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