WO2017009926A1 - Fixing method for specific binding substance - Google Patents

Fixing method for specific binding substance 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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French (fr)
Japanese (ja)
Inventor
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/en
Publication of WO2017009926A1 publication Critical patent/WO2017009926A1/en

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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”.

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Abstract

The present invention addresses the problem of providing a fixing method for a specific binding substance, capable of suppressing a phenomenon in which a positive line emerges even in a blank measurement in an immunotrap method, and providing a test device (carrier) capable of suppressing emergence of a positive line. The present invention pertains to: a fixing method for a specific binding substance, characterized by premixing a substance (specific binding substance) which specifically binds to a substance to be measured and a membrane solubilizing agent selected from the group consisting of n-octyl-β-D-glucoside, n-octyl-β-D-thioglucoside, n-dodecyl-β-D-maltoside, n-nonyl-β-D-thiomaltoside, and n-octanoyl-N-methyl-D-glucamine, and applying, onto a surface of a nonabsorptive carrier, a mixture liquid containing the specific binding substance and the membrane solubilizing agent, thereby fixing the specific binding substance to the surface; a nonabsorptive carrier obtained through the fixing method; and a measuring method for a substance to be measured in a sample, characterized by using the nonabsorptive carrier.

Description

特異的結合物質の固定化方法Method for immobilizing specific binding substances
　本発明は、測定対象物質に特異的に結合する物質（以下、特異的結合物質と略記する場合がある。）を非吸収性担体の表面に固定化させる方法等に関する。さらに詳しくは、特異的結合物質と特定の膜可溶化剤とをあらかじめ混合し、該特異的結合物質と該膜可溶化剤とを含む混合液を非吸収性担体表面に塗布することにより、特異的結合物質を非吸収性担体の表面に固定化させる固定化方法、該固定化方法により得られる非吸収性担体、ならびに該非吸収性担体を用いることを特徴とする、試料中の測定対象物質の測定方法に関する。 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.
　イムノクロマトグラフィー法（以下、イムノクロマト法と略記する場合がある。）は、金コロイドや着色ラテックス等のナノ粒子と結合した被検体がニトロセルロース等の膜上を毛細管現象により展開することを利用した診断手法である。この手法は操作が簡便で、判定までに要する時間が１０～３０分程度と比較的短時間であり、判定に高価な装置を要しないことから優れた簡易診断手法として臨床の現場で多用されている。例えばインフルエンザウイルスやＲＳウイルス等のウイルスの感染の判定においては、患者から採取した鼻腔ぬぐい液や咽頭ぬぐい液を検体として、短時間かつその場での判定が可能であり、感染の簡易診断手法として非常に有力なツールとして普及している。 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.
　イムノクロマト法では一般的に金コロイドや着色ラテックス等のナノ粒子が標識粒子として用いられ、被検物質に対する抗体等の特異的結合物質が感作された標識粒子と被検物質が不溶性薄膜状支持体であるニトロセルロース等の膜上を毛細管現象により展開し、不溶性薄膜状支持体にあらかじめ固定化されている被検物質に対する抗体等の特異的結合物質と結合することで、特異的結合物質感作標識粒子－被検物質－特異的結合物質固定化不溶性薄膜状支持体の複合体を形成し、陽性ラインを得る。しかしながら、上述したようなナノ粒子を用いたイムノクロマト法でも、陰性を判定するためには１０分以上の反応時間を要する。 In immunochromatography, 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. 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. However, even the immunochromatography method using nanoparticles as described above requires a reaction time of 10 minutes or more in order to determine negative.
　一方、特許文献１では、水浸透性を有しない担体上に被検物質に対する特異的結合物質を部分的に被覆し、該担体の表面に試料を接触させた後、その試料を除去して、上記担体の表面上に被覆されている特異的結合物質と同一または異なる特異的結合物質を固定化した粒子を上記面に接触させ、担体上を移動させることで、イムノクロマト法よりも短時間での診断を可能にしている。この技術を応用した非特許文献１に記載のイムノトラップ法は、プラスチック基板上の判定部にあらかじめ測定対象物質に対する特異的結合物質を塗布しておき、測定対象物質を含んだ試料をプラスチック基板上に移動させた後、測定対象物質と同一または異なる特異的結合物質を感作させた磁性粒子を磁力によりプラスチック基板上を泳動させることで、陽性の場合、判定部にラインが生じるというものであり、陽性または陰性の結果判定が判定時間１分で可能となる。 On the other hand, in 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. In the immunotrap method described in Non-Patent Document 1 to which this technology is applied, 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. After moving to, 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.
　しかしながら、イムノクロマト法や上述したイムノトラップ法等では、抗原抗体反応等の測定対象物質と特異的に結合する反応を利用しているにもかかわらず、非特異的な結合を回避するための洗浄工程を行わないため、非特異的な反応が生じて、測定対象物質を含まない試料でも非特異的なラインを発現しやすいという問題点がある。 However, in the immunochromatography method and the immunotrap method described above, a washing step for avoiding non-specific binding despite using a reaction that specifically binds to a substance to be measured such as an antigen-antibody reaction. Therefore, there is a problem that a non-specific reaction occurs and a non-specific line is easily developed even in a sample that does not contain a measurement target substance.
特開平１０－２２７７９４号公報JP-A-10-227794
　イムノトラップ法において、プラスチック基板上に抗体等を塗布する場合、通常の塗布方法では抗体等の溶液がプレート内に浸み込まず、ラインを形成することができない。そのため、単純な緩衝液中に抗体を溶解させて塗布すると、ブランク測定においても陽性ライン（非特異ライン）が発現してしまうという問題が生じることがわかった。そこで、プラスチック担体等を用いた場合でも、このような非特異ラインの発現を抑制できる特異的結合物質の固定化方法、ならびに非特異ラインの発現を抑制できるテストデバイス（担体）の開発が望まれている。 In the immuno trap method, when an antibody or the like is applied on a plastic substrate, a solution such as an antibody does not soak into the plate and a line cannot be formed by a normal application method. Therefore, it was found that when the antibody was dissolved and applied in a simple buffer solution, a positive line (non-specific line) was developed even in the blank measurement. Therefore, it is desired to develop a method for immobilizing a specific binding substance that can suppress the expression of such non-specific lines and a test device (carrier) that can suppress the expression of non-specific lines even when using a plastic carrier or the like. ing.
　本発明は、上述した状況に鑑みなされたものであり、イムノトラップ法において非特異ラインの発現を抑制できる特異的結合物質の固定化方法を提供すること、ならびに非特異ラインの発現を抑制できるテストデバイス（担体）を提供することにある。 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).
　本発明は、測定対象物質に特異的に結合する物質（特異的結合物質）と、ｎ-オクチル-β-Ｄ-グルコシド、ｎ-オクチル-β-Ｄ-チオグルコシド、ｎ-ドデシル-β-Ｄ-マルトシド、ｎ-ノニル-β-Ｄ-チオマルトシドおよびｎ-オクタノイル-Ｎ-メチル-Ｄ-グルカミンからなる群から選ばれる膜可溶化剤とをあらかじめ混合し、該特異的結合物質と該膜可溶化剤とを含む混合液を非吸収性担体の表面に塗布して、該表面に特異的結合物質を固定化することを特徴とする、特異的結合物質の固定化方法の発明である。 The present invention relates to a substance that specifically binds to a substance to be measured (specific binding substance), n-octyl-β-D-glucoside, n-octyl-β-D-thioglucoside, n-dodecyl-β-D. A membrane solubilizer selected from the group consisting of -maltoside, n-nonyl-β-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.
　また、本発明は、測定対象物質に特異的に結合する物質（特異的結合物質）と、ｎ-オクチル-β-Ｄ-グルコシド、ｎ-オクチル-β-Ｄ-チオグルコシド、ｎ-ドデシル-β-Ｄ-マルトシド、ｎ-ノニル-β-Ｄ-チオマルトシドおよびｎ-オクタノイル-Ｎ-メチル-Ｄ-グルカミンからなる群から選ばれる膜可溶化剤とをあらかじめ混合し、該特異的結合物質と該膜可溶化剤とを含む混合液を非吸収性担体の表面に塗布することによって得られる、該表面に特異的結合物質が固定化された非吸収性担体の発明である。 Further, the present invention relates to a substance that specifically binds to a substance to be measured (specific binding substance), n-octyl-β-D-glucoside, n-octyl-β-D-thioglucoside, n-dodecyl-β. A membrane solubilizer selected from the group consisting of -D-maltoside, n-nonyl-β-D-thiomaltoside and n-octanoyl-N-methyl-D-glucamine is mixed in advance, and the specific binding substance and the membrane It is an invention of a non-absorbable carrier obtained by applying a mixed solution containing a solubilizer to the surface of the non-absorbable carrier and having a specific binding substance immobilized on the surface.
　さらに、本発明は、測定対象物質に特異的に結合する物質（特異的結合物質）と、ｎ-オクチル-β-Ｄ-グルコシド、ｎ-オクチル-β-Ｄ-チオグルコシド、ｎ-ドデシル-β-Ｄ-マルトシド、ｎ-ノニル-β-Ｄ-チオマルトシドおよびｎ-オクタノイル-Ｎ-メチル-Ｄ-グルカミンからなる群から選ばれる膜可溶化剤とをあらかじめ混合し、該特異的結合物質と該膜可溶化剤とを含む混合液を非吸収性担体の表面に塗布することによって得られる、該表面に特異的結合物質が固定化された非吸収性担体を用いることを特徴とする、試料中の測定対象物質の測定方法の発明である。 Furthermore, the present invention relates to a substance that specifically binds to a substance to be measured (specific binding substance), n-octyl-β-D-glucoside, n-octyl-β-D-thioglucoside, n-dodecyl-β. A membrane solubilizer selected from the group consisting of -D-maltoside, n-nonyl-β-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.
　本発明の特異的結合物質の固定化方法を用いて得られた非吸収性担体を、例えばインフルエンザウイルス、ＲＳウイルス等のウイルスの測定に用いることにより、非特異反応を抑制でき、より精度の高い判定ができる。 By using a 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.
各ウェルの平底面の片側半分が特異的結合物質により被覆されている平底マイクロプレートを示す図である。It is a figure which shows the flat bottom microplate by which one half of the flat bottom face of each well is coat | covered with the specific binding substance. 底面が特異的結合物質により帯状に被覆されている直方体型の透明容器を示す図である。It is a figure which shows the rectangular parallelepiped type transparent container by which the bottom face is coat | covered with the specific binding substance in the strip | belt shape. 表面が特異的結合物質により帯状に被覆されている板状体を示す図である。It is a figure which shows the plate-shaped body by which the surface is coat | covered with the specific binding substance in the strip | belt shape. 表面に流路（溝）が設けられ、該流路の底面が特異的結合物質により帯状に被覆されている板状体を示す図である。It is a figure which shows the plate-shaped object by which the flow path (groove | channel) was provided in the surface and the bottom face of this flow path was coat | covered with the specific binding substance in the strip | belt shape. 表面に流路（溝）が設けられ、該流路の底面が特異的結合物質により帯状に被覆されている板状体を示す図である。It is a figure which shows the plate-shaped object by which the flow path (groove | channel) was provided in the surface and the bottom face of this flow path was coat | covered with the specific binding substance in the strip | belt shape.
　本発明は、測定対象物質に特異的に結合する物質（特異的結合物質）と、ｎ-オクチル-β-Ｄ-グルコシド、ｎ-オクチル-β-Ｄ-チオグルコシド、ｎ-ドデシル-β-Ｄ-マルトシド、ｎ-ノニル-β-Ｄ-チオマルトシドおよびｎ-オクタノイル-Ｎ-メチル-Ｄ-グルカミンからなる群から選ばれる膜可溶化剤（以下、本発明に係る膜可溶化剤と略記する場合がある。）とをあらかじめ混合し、該特異的結合物質と該膜可溶化剤とを含む混合液を非吸収性担体の表面に塗布して、該表面に特異的結合物質を固定化することを特徴とする、特異的結合物質の固定化方法の発明である。 The present invention relates to a substance that specifically binds to a substance to be measured (specific binding substance), n-octyl-β-D-glucoside, n-octyl-β-D-thioglucoside, n-dodecyl-β-D. Membrane solubilizer selected from the group consisting of -maltoside, n-nonyl-β-D-thiomaltoside and n-octanoyl-N-methyl-D-glucamine (hereinafter sometimes abbreviated as membrane solubilizer according to the present invention). And 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.
　また、本発明は、測定対象物質に特異的に結合する物質（特異的結合物質）と、本発明に係る膜可溶化剤とをあらかじめ混合し、該特異的結合物質と該膜可溶化剤とを含む混合液を非吸収性担体の表面に塗布することによって得られる、該表面に特異的結合物質が固定化された非吸収性担体の発明である。 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.
　さらに、本発明は、測定対象物質に特異的に結合する物質（特異的結合物質）と、本発明に係る膜可溶化剤とをあらかじめ混合し、該特異的結合物質と該膜可溶化剤とを含む混合液を非吸収性担体の表面に塗布することによって得られる、該表面に特異的結合物質が固定化された非吸収性担体を用いることを特徴とする、試料中の測定対象物質の測定方法の発明である。 Furthermore, 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.
　本発明に係る測定対象物質としては、例えばタンパク質、糖質、脂質、核酸のような有機物質、無機物質等の生体関連物質であればいずれのものでもよい。測定対象物質の具体例としては、例えばＨＢｓ抗原、抗ＨＢｓ抗体、ＨＢｅ抗原、抗ＨＢｅ抗体、抗ＨＢｃ抗体、抗ＨＣＶ抗体、抗ＨＩＶ抗体、抗ＡＴＬＶ抗体、インフルエンザ抗原、抗インフルエンザ抗体、ＲＳウイルス抗原、抗ＲＳウイルス抗体、アデノウイルス抗原、抗アデノウイルス抗体、ヒトメタニューモウイルス抗原、ノロウイルス抗原等のウイルス関連の抗原または抗体；例えば大腸菌Ｏ１５７抗原、抗トレポネーマ・パリダム（ＴＰ）抗体、抗カルジオリピン抗体、マイコプラズマ抗原、抗マイコプラズマ抗体、Ａ群溶血性レンサ球菌抗原、抗ストレプトリジンＯ抗体（ＡＳＯ）等の細菌関連の抗原または抗体；例えば免疫グロブリンＧ（ＩｇＧ）、免疫グロブリンＡ（ＩｇＡ）、免疫グロブリンＭ（ＩｇＭ）、免疫グロブリンＥ（ＩｇＥ）等の免疫グロブリン；例えばＣ反応性タンパク質（ＣＲＰ）、α１-酸性糖タンパク質、ハプトグロビン、補体Ｃ３、補体Ｃ４、リウマトイド因子等の炎症マーカー；例えばα-フェトプロテイン、ＣＥＡ、ＣＡ１９-９等の腫瘍マーカー；例えばヒト胎盤絨毛性ゴナドトロピン等のホルモン；例えばアレルゲン、アレルゲン特異ＩｇＥ抗体等のアレルギー関連の抗原または抗体；例えば抗トロンビンＩＩＩ（ＡＴＩＩＩ）等の血液凝固系関連物質；例えばフィブリン体分解物（ＦＤＰ）、Ｄダイマー等の線溶系関連物質；例えばＡＢＯ式血液型抗体、不規則抗体等の血液型関連の抗原または抗体；例えばウイルスのＤＮＡまたはＲＮＡ；例えば細菌のＤＮＡまたはＲＮＡ；例えばヒト等の動物もしくは植物のＤＮＡまたはＲＮＡ；リポタンパク質（ａ）等の他の疾病に関連した物質または薬物等が挙げられ、なかでも、インフルエンザ抗原、ＲＳウイルス抗原、アデノウイルス抗原、ヒトメタニューモウイルス抗原、ノロウイルス抗原、マイコプラズマ抗原、Ａ群溶血性レンサ球菌抗原が好ましく、そのなかでも、インフルエンザ抗原、ＲＳウイルス抗原がより好ましい。 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. Specific examples of 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), α1-acid glycoprotein, haptoglobin, complement C3, complement C4, rheumatoid factor; for example α-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 (FDP), D-dimer and the like; blood group-related antigens or antibodies such as ABO blood group antibodies and irregular antibodies; for example, viral DNA or RNA; for example, bacterial DNA or RNA; For example, DNA or RNA of animals such as humans or plants Examples include substances or drugs related to other diseases such as lipoprotein (a), among which influenza antigen, RS virus antigen, adenovirus antigen, human metapneumovirus antigen, norovirus antigen, mycoplasma antigen, group A hemolysis Sexual streptococcal antigens are preferred, and among them, influenza antigens and RS virus antigens are more preferred.
　本発明に係る、測定対象物質に特異的に結合する物質（以下、特異的結合物質と略記する場合がある。）とは、測定対象物質に対し親和性を有する物質をいい、例えば蛋白質－蛋白質相互作用、蛋白質－化学物質相互作用、化学物質－化学物質相互作用に応じて目的物質と結合することが可能な性質を有するものが挙げられる。具体的には、抗原－抗体相互作用、糖鎖－レクチン相互作用、酵素－インヒビター相互作用、蛋白質－ペプチド鎖相互作用、染色体もしくは核酸鎖－核酸鎖相互作用、ヌクレオチド－リガンド相互作用、受容体－リガンド相互作用に基づいて結合するものが挙げられる。担体に固定化される特異的結合物質は、測定対象物質を介しての結合が可能であれば、それぞれ同一でも異なってもよい。 The substance that specifically binds to the substance to be measured according to the present invention (hereinafter sometimes abbreviated as a specific binding substance) 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.
　本発明に係る膜可溶化剤は、ｎ-オクチル-β-Ｄ-グルコシド、ｎ-オクチル-β-Ｄ-チオグルコシド、ｎ-ドデシル-β-Ｄ-マルトシド、ｎ-ノニル-β-Ｄ-チオマルトシドおよびｎ-オクタノイル-Ｎ-メチル-Ｄ-グルカミンから選ばれる界面活性剤である。これらの膜可溶化剤は、非特異反応を抑制するためのものであり、本発明においては、特定の膜可溶化剤を用いることにより、非特異反応を抑制できる。さらに、本発明においては、これらの膜可溶化剤を特異的結合物質とあらかじめ混合し、この混合液を後述する担体に塗布することによってのみ、非特異反応を抑制できる。 The membrane solubilizing agent according to the present invention includes n-octyl-β-D-glucoside, n-octyl-β-D-thioglucoside, n-dodecyl-β-D-maltoside, n-nonyl-β-D-thiomaltoside. And a surfactant selected from n-octanoyl-N-methyl-D-glucamine. These membrane solubilizers are for suppressing non-specific reactions. In the present invention, non-specific reactions can be suppressed by using specific membrane solubilizers. Furthermore, in the present invention, 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.
　本発明に係る非吸収性担体は、非浸透性の担体、すなわち、特異的結合物質と膜可溶化剤とを含む溶液や試料を含む溶液（例えば試料や試料希釈液等）が、特異的結合物質が被覆される面から担体の内部に浸透しない、非浸透性のものであることが望ましい。 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.
　本発明に係る非吸収性担体の材質は、特異的結合物質を物理的または化学的に固定化でき、なおかつ上述したような非浸透性のものであれば何ら制限されない。このような非吸収性担体の材質としては、例えばポリエチレン、ポリプロピレン、ポリスチレン、ポリ塩化ビニル、ポリアクリレート、ポリメタクリレート、ポリカーボネートまたはナイロン製のプラスチック等が挙げられる。 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.
　なお、本発明に係る非吸収性担体には、例えばセルロース等の吸収性担体（浸透性の担体）の表面に上述したプラスチック（シート）等の非吸収性担体を接着させたものや、吸収性担体（浸透性の担体）の表面にプラスチック等の非吸収性担体を積層させたもの等も含まれる。 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).
　本発明に係る非吸収性担体としては、特異的結合物質を固定化することにより該特異的結合物質が部分的に被覆（担持）された面、すなわち、特異的結合物質によって被覆されていない部分と被覆されている部分を併せ持つ面を有する非吸収性担体を使用してもよいし、該特異的結合物質が全面的に被覆（担持）された面、すなわち、特異的結合物質によって被覆されている部分のみを持つ面を有する非吸収性担体を使用してもよい。このような非吸収性担体のなかでも、該特異的結合物質が部分的に被覆（担持）された面、すなわち、特異的結合物質によって被覆されていない部分と被覆されている部分を併せ持つ面を有する非吸収性担体を使用することが好ましい。担体としては特異的結合物質により全面的または部分的に被覆されている面を有しており、該面に試料等の溶液（液体）を接触させることができる限りいずれの形状、構造でもよく、例えば容器、板状体等が挙げられる。 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. Among such non-absorbable carriers, 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. For example, a container, a plate-shaped body, etc. are mentioned.
　本発明の非吸収性担体として容器状のものを用いる場合、溶液収納部分である凹部の形状は半球状、円筒形状、直方体形状等いずれの形状でもよい。また、その凹部の内壁面を特異的結合物質により全面的または部分的に被覆すればよく、なかでも、部分的に被覆することが好ましい。凹部の形状が円筒形状、直方体形状等の平底面を有する形状である場合には、その平底面を特異的結合物質により全面的または部分的に被覆すればよく、なかでも、部分的に被覆することが好ましい。容器は凹部が複数存在していてもよい。容器としては、平底面を有する凹部を少なくとも一つ備えた容器であって、その平底面が特異的結合物質により部分的に被覆することが好ましい。平底面を有する凹部を少なくとも一つ備えた容器としては、例えば平底マイクロプレート、トレイ等が挙げられる。 In the case of using a container as the non-absorbable carrier of the present invention, 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. Further, 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. When 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.
　本発明の非吸収性担体として板状体のものを使用する場合、板状体の表面形状は円形、長方形、正方形等いずれの形状であってもよく、その表面を特異的結合物質により全面的または部分的に被覆すればよい。また、板状体の表面に溝または壁を形成することにより流路を形成させ、該流路に試料等の溶液を導入することにより溶液が表面から流れ落ちるのを防ぐこともできる。 When a plate-like body is used as the non-absorbable carrier of the present invention, 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.
　本発明において、特異的結合物質と膜可溶化剤を混合する方法としては、通常この分野で一般的に行われている方法であれば特に制限されず、例えばリン酸緩衝生理食塩水（ＰＢＳ）、トリス（ヒドロキシメチル）アミノメタン緩衝液、グッド緩衝液、ホウ酸緩衝液等の緩衝液中、特定濃度の膜可溶化剤と特異的結合物質とを混合し、特定濃度の溶液にする方法が挙げられ、具体的には、例えば膜可溶化剤を緩衝液に溶解させた溶液に、特異的結合物質を添加する方法、特異的結合物質を緩衝液に溶解させた溶液に、膜可溶化剤を添加する方法、膜可溶化剤を緩衝液に溶解させた溶液と特異的結合物質を緩衝液に溶解させた溶液とを混合する方法等が挙げられる。なお、上述した緩衝液のｐＨについては、４～１２の範囲内にあることが好ましい。 In the present invention, 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). In 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.
　特異的結合物質と膜可溶化剤を混合した後の、特異的結合物質と膜可溶化剤とを含む混合液中の特異的結合物質の濃度としては、通常０．０１～１０ｍｇ／ｍＬ、好ましくは０．１～５ｍｇ／ｍＬである。 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.
　特異的結合物質と膜可溶化剤を混合した後の、特異的結合物質と膜可溶化剤とを含む混合液中の膜可溶化剤の濃度としては、通常０．００１～１％（ｗ／ｖｏｌ）、好ましくは０．００１～０．１％（ｗ／ｖｏｌ）である。 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).
　特異的結合物質の非吸収性担体の表面への固定化は、疎水結合、親水吸着等の物理的吸着法または架橋試薬による化学的結合法によって行うことができる。該固定化を物理的吸着法によって行う場合には、公知の方法に従い、例えば特異的結合物質および本発明に係る膜可溶化剤を含む混合液を、非吸収性担体の面に接触させることにより行うことができる。例えば非吸収性担体が容器の場合、特異的結合物質および本発明に係る膜可溶化剤を含む混合液を該容器の凹部に入れて静置することにより、特異的結合物質を担体に接触させ、約２～４０℃で約１０分～１日間吸着反応を行わせた後、凹部の液を吸引除去し、緩衝液等で洗浄することにより、特異的結合物質を非吸収性担体に固定化させることができる。 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. When 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. For example, when 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. After 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.
　また、該固定化を架橋試薬による化学的結合法によって行う場合には、日本臨床病理学会編「臨床病理臨時増刊特集第５３号 臨床検査のためのイムノアッセイ－技術と応用－」、臨床病理刊行会、１９８３年、日本生化学会編「新生化学実験講座１ タンパク質IV」、東京化学同人、１９９１年等に記載の公知の方法に従い、特異的結合物質および本発明に係る膜可溶化剤を含む混合液と非吸収性担体を接触させる前か、または同時にグルタルアルデヒド、カルボジイミド、イミドエステル、マレイミド等の二価性の架橋試薬を非吸収性担体に接触させ、特異的結合物質と非吸収性担体のそれぞれのアミノ基、カルボキシル基、チオール基、アルデヒド基、水酸基等とを反応させることにより、特異的結合物質を非吸収性担体に固定化させることができる。例えば非吸収性担体が容器の場合、該容器の凹部にグルタルアルデヒド、カルボジイミド、イミドエステル、マレイミド等の二価性の架橋試薬を加え、静置して反応させる。次いで、これに特異的結合物質および本発明に係る膜可溶化剤を含む混合液を加えて静置することにより反応させる。場合によっては、その後、これに架橋反応の反応停止剤を添加すること等により反応を停止させる。そして、緩衝液等で洗浄することにより、特異的結合物質を非吸収性担体に固定化させることができる。 When the immobilization is performed by a chemical binding method using a cross-linking reagent, the Japanese Society of Clinical Pathology “Special Issue on Clinical Pathology No. 53 Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publication Society In 1983, according to a known method described in the “Shinsei Chemistry Experiment Course 1 Protein IV” edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1991, etc., a mixed solution containing a specific binding substance and the membrane solubilizing agent according to the present invention Before contacting the non-absorbable carrier with a non-absorbable carrier, or at the same time, bringing a bivalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, maleimide, etc. into contact with the 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. It is possible. For example, when the non-absorbable carrier is a container, 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. Next, 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. In some cases, 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.
　非吸収性担体の表面を部分的に被覆する方法としては、例えば該表面の特異的結合物質で被覆したい部分にのみ特異的結合物質と膜可溶化剤とをあらかじめ混合することによって得られた混合液または該混合液と架橋試薬とを滴下して上記方法により固定化する方法、表面の特異的結合物質で被覆したい部分の周囲を例えばプラスチック板等の非吸収性の材料で囲って、この囲まれた部分に特異的結合物質と膜可溶化剤とをあらかじめ混合することによって得られた混合液または該混合液と架橋試薬とを滴下して上記方法により固定化する方法、スポンジ等の吸収体に特異的結合物質と膜可溶化剤とをあらかじめ混合することによって得られた混合液または該混合液と架橋試薬とを吸収させ、面の特異的結合物質で被覆したい部分に置いて接触させるかまたは塗布して上記方法により固定化する方法、あるいは特異的結合物質と膜可溶化剤とをあらかじめ混合することによって得られた混合液をパルスインジェクター（登録商標）等の塗布装置を用いて塗布し、上記方法により固定化する方法等が挙げられる。 As 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 method in which a solution or a mixed solution and a crosslinking reagent are dropped and immobilized by the above-described method, and a portion of a surface to be coated with a specific binding substance is surrounded by a non-absorbable material such as a plastic plate. A mixture obtained by mixing a specific binding substance and a membrane solubilizing agent in advance in the part, or a method in which the mixture and the crosslinking reagent are dropped and immobilized by the above method, an absorbent such as a sponge Absorb the mixed solution obtained by previously mixing the specific binding substance and the membrane solubilizer or the mixed liquid and the crosslinking reagent, and place it on the surface to be coated with the specific binding substance. Using a coating device such as Pulse Injector (registered trademark), a method of touching or coating and immobilizing by the above method, or a mixture obtained by mixing a specific binding substance and a membrane solubilizer in advance. The method of apply | coating and fixing by the said method etc. are mentioned.
　本発明において、特異的結合物質と膜可溶化剤とをあらかじめ混合することによって得られた混合液または該混合液と架橋試薬とを非吸収性担体に塗布する方法としては、通常この分野で一般的に行われている方法であれば特に制限されず、本発明の固定化方法においては、パルスインジェクター（登録商標）等の塗布装置を用いて、特異的結合物質と膜可溶化剤とをあらかじめ混合することによって得られた混合液または該混合液と架橋試薬とを非吸収性担体に塗布することが好ましい。パルスインジェクター（登録商標）等の塗布装置を用いて塗布する方法の具体例としては、例えば特開２００２－１７６２０５号公報に記載の方法、すなわち、小型の圧電素子を液体射出ヘッドに組み込んだ塗布装置の液体射出ヘッドを利用して粘性の高い液体を精度よく射出することにより塗布する方法等が挙げられる。より具体的には、例えば特異的結合物質と膜可溶化剤とをあらかじめ混合することによって得られた混合液または該混合液と架橋試薬とを塗布装置の液体射出ヘッドに充填した後、非吸収性担体上の塗布する部分の上側に液体射出ヘッドをセットし、混合液または混合液と架橋試薬とを３～３０ｐＬ／滴で連続吐出する。塗布された混合液または混合液と架橋試薬は短時間に自然乾燥し、特異的結合物質が非吸収性担体上に固定化される。 In the present invention, 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. In the immobilization method of the present invention, 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. As a specific example of 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 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.
　さらに、必要であれば非特異的反応を抑制するため、ウシ血清アルブミン、ヒト血清アルブミン、カゼインまたはその塩等の各種タンパク質、脱脂粉乳等を、特異的結合物質が固定化された担体に接触させること等の公知の方法により、特異的結合物質が固定化された担体をブロッキングしてもよい。ブロッキング液としては、具体的には、例えばウシアルブミン、カゼイン等の蛋白成分を１．０～５０ｍｇ／ｍＬ含んだリン酸緩衝生理食塩水（ＰＢＳ）、トリス（ヒドロキシメチル）アミノメタン緩衝液、グッド緩衝液等が用いられる。なお、上述した緩衝液のｐＨについては、４～１２の範囲内にあることが好ましい。ブロッキング方法としては、例えば特異的結合物質を固定化した非吸収性担体を上述したブロッキング液に浸漬し、１～４０℃で１０分～７２時間静置する方法等が挙げられる。ブロッキング後は安定化のために減圧乾燥を行ってもよい。減圧乾燥方法としては、例えば０．１～１０Ｐａの減圧下で１～７２時間静置する方法等が挙げられる。 Furthermore, in order to suppress non-specific reactions if necessary, 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. 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.
　本発明の固定化方法によって得られた非吸収性担体は、例えば上述のイムノトラップ法における担体として用いることができる。より具体的には、当該非吸収性担体の表面に、試料を接触させた後、測定対象物質に対する特異的結合物質であって上記非吸収性担体に固定化された特異的結合物質と同一または異なる特異的結合物質が固定化された粒子とを接触させ、当該粒子が上記表面に沿って移動するように該担体を移動させて、上記表面における粒子の分布状態から測定対象物質の有無を判定する方法の担体として用いることができる。 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.
　上述した粒子としては、一般に間接凝集反応に用いられる粒子が挙げられる。例えばリポソ－ム、ラテックス粒子、ゼラチン粒子、ポリアクリルアミド粒子、マイクロカプセル、エマルジョン等の有機高分子粒子、ガラスビ－ズ、シリカビ－ズ、ベントナイト等の無機高分子粒子、他の人工粒子、赤血球等が挙げられる。 As the above-mentioned particles, there are particles generally used for indirect agglutination reaction. For example, 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. Can be mentioned.
　また、粒子として磁性粒子を用いることもできる。磁性粒子は、少なくとも外部から磁石を作用させている間は磁化する粒子であればよい。当該磁性粒子としては、例えば鉄、コバルト、ニッケル等の強磁性金属、これらの強磁性金属を含む合金、非磁性体中に強磁性金属または強磁性金属を含む合金を含有するもの、強磁性金属中または強磁性金属を含む合金中に非磁性体を含有するもの等の強磁性体を単独で粒子状に成形した粒子、強磁性体を核としてその表面をポリスチレン、シリカゲル、ゼラチン、ポリアクリルアミド等の高分子物質で被覆した粒子、ポリスチレン、シリカゲル、ゼラチン、ポリアクリルアミド等の高分子物質の粒子を核として強磁性体を被覆した粒子、赤血球、リポソームまたはマイクロカプセル等の閉じた袋状の物質に強磁性体を封入した粒子等が挙げられる。なお、該磁性粒子は、外部から磁石を作用させている間は磁化し、外部からの磁石の遮断により速やかに減磁する性質を持つものであることが特に好ましく、そのような磁性粒子としては、例えば強磁性体である酸化鉄（III）（Ｆｅ２Ｏ３）を粒子内に分散させた磁性粒子である「Dynabeads（登録商標） M-450 uncoated（商品名）（ライフテクノロジーズ社製）」、「Dynabeads（登録商標） M-450 Epoxy（商品名）（ライフテクノロジーズ社製）」等が挙げられる。 Also, 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. Examples of 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 in which a ferromagnetic material such as one containing a non-magnetic material is contained inside or in an alloy containing a ferromagnetic metal, and the surface of the ferromagnetic material is polystyrene, silica gel, gelatin, polyacrylamide, etc. 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. In addition, it is particularly preferable that 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. For example, “Dynabeads (registered trademark) M-450 uncoated (trade name) (manufactured by Life Technologies)” which is a magnetic particle in which iron (III) (Fe 2 O 3 ), which is a ferromagnetic material, is dispersed in the particle. , “Dynabeads (registered trademark) M-450 Epoxy (trade name) (manufactured by Life Technologies)” and the like.
　さらに、粒子としては、色素を被覆するかまたは色素を粒子中に分散もしくは封入させることにより着色したものを使用してもよく、また、色素は蛍光色素であってもよい。 Further, as the particles, 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.
　粒子の粒子径は、通常０．００１～１０００μｍ、好ましくは０．０１～１００μｍ、より好ましくは０．５～１０μｍである。また、粒子の比重は、分散媒中で沈降する比重であればよく、例えば比重１～１０のものが好ましい。 The particle diameter of the particles is usually 0.001 to 1000 μm, preferably 0.01 to 100 μm, more preferably 0.5 to 10 μm. 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.
　本発明の非吸収性担体を用いて測定を行う場合には、特異的結合物質を固定化した粒子を用いるが、特異的結合物質を粒子に固定化するには、特異的結合物質を上述した粒子の表面に、疎水結合、親水吸着等の物理的吸着法、共有結合等の化学的結合法またはこれらの方法の併用等により行うことができる。 When measurement is performed using the non-absorbable carrier of the present invention, particles having a specific binding substance immobilized thereon are used. In order to immobilize a specific binding substance on the particles, 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.
　特異的結合物質の粒子への固定化を物理的吸着法により行う場合は、公知の方法に従って、該特異的結合物質と粒子とを緩衝液等の溶液中で混合し接触させることにより行うことができる。例えば特異的結合物質と粒子を緩衝液等の溶液中で混合し撹拌することにより接触させ、約２～４０℃で約１０分～１日間吸着反応を行わせた後、得られた粒子を緩衝液等で洗浄すること等により、特異的結合物質を粒子に固定化することができる。 When the specific binding substance is immobilized on the particles by a physical adsorption method, 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. For example, 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.
　また、特異的結合物質の粒子への固定化を架橋試薬による化学的結合法によって行う場合は、日本臨床病理学会編「臨床病理臨時増刊特集第５３号 臨床検査のためのイムノアッセイ－技術と応用－」、臨床病理刊行会、１９８３年、日本生化学会編「新生化学実験講座１ タンパク質IV」、東京化学同人、１９９１年等に記載の公知の方法に従って、特異的結合物質と粒子をグルタルアルデヒド、カルボジイミド、イミドエステル、マレイミド等の二価性の架橋試薬と混合、接触させ、特異的結合物質と粒子のそれぞれのアミノ基、カルボキシル基、チオール基、アルデヒド基、水酸基等の官能基を架橋試薬と反応させることにより、特異的結合物質を粒子に固定化することができる。例えば粒子を含む緩衝液等にグルタルアルデヒド、カルボジイミド、イミドエステル、マレイミド等の二価性の架橋試薬を加え、撹拌し反応させる。次いで、これに特異的結合物質を加え、撹拌して反応させる。場合によっては、その後、これに透析、ゲルろ過等の処理により架橋試薬を除くかもしくは架橋反応の反応停止剤を添加すること等により反応を停止させる。そして、得られた粒子を緩衝液等で洗浄すること等により、特異的結合物質を粒子に固定化することができる。 In addition, when the specific binding substance is immobilized on the particles by a chemical binding method using a cross-linking reagent, the Clinical Pathology Special Edition No. 53 Special Issue on Clinical Pathology-Immunoassay for Clinical Examination -Technology and Applications- In accordance with a known method described in “Clinical Pathology Publishing Society, 1983”, “Nippon Kagaku Kenkyu Koza 1” Protein IV ”, Tokyo Kagaku Dojin, 1991, etc., edited by the Japanese Biochemical Society, glutaraldehyde, carbodiimide , Imidoesters, maleimides, and other bivalent cross-linking reagents, and contact with them to react specific binding substances and functional groups such as amino groups, carboxyl groups, thiol groups, aldehyde groups, and hydroxyl groups with the cross-linking reagents. By doing so, the specific binding substance can be immobilized on the particles. For example, 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.
　また、特異的結合物質の粒子への固定量を変更することにより、試料中の測定対象物質の濃度の高低に応じて容易に感度を変更することができる。例えば粒子に特異的結合物質を固定化する際に、高濃度の特異的結合物質を用いれば固定量が多くなって感度を高めることができる。 Also, by changing the amount of specific binding substance immobilized on the particles, 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.
　必要であれば非特異的反応を抑制するために、ウシ血清アルブミン、ヒト血清アルブミン、カゼインまたはその塩等の各種タンパク質、脱脂粉乳等を、特異的結合物質が固定化された粒子に接触させること等の公知の方法により、該粒子をマスキングしてもよい。マスキングは、例えば特異的結合物質が固定化された粒子をウシ血清アルブミン、ヒト血清アルブミン、カゼインまたはその塩等の各種タンパク質等を含む緩衝液等に加え静置し、該粒子の表面を各種タンパク質等でコーティングすることにより行うことができる。 If necessary, 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.
　上述した試料とは、上述した測定対象物質が存在する可能性があり、かつその測定対象物質の存在の有無の確認または場合によっては定量を行おうとする液状のものをいう。例えばヒトまたは動物の血液、血清、血漿、尿、精液、髄液、唾液、汗、涙、腹水、羊水等の体液；例えばヒトもしくは動物の脳等の臓器、毛髪、皮膚、爪、筋肉、または神経組織等の抽出液；例えばヒトまたは動物の糞便の抽出液または懸濁液；細胞あるいは菌体の抽出液；植物の抽出液等が挙げられる。 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.
　本発明の非吸収性担体を用いた測定方法としては、例えば上述したように特異的結合物質を固定化し、特異的結合物質で部分的に被覆されている面、すなわち、特異的結合物質によって被覆されていない部分と被覆されている部分を併せ持つ面を有する担体の表面に、測定対象物質の存在が疑われる試料を接触させる方法が挙げられる（以下、本発明の第１の測定方法と略記する場合がある。）。 As a measurement method using the non-absorbable carrier of the present invention, for example, as described above, 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.)
　非吸収性担体が例えば上述したような容器の場合には、該容器の凹部に試料を添加することにより容器の内壁面と試料とを接触させることができる。また、非吸収性担体が、例えば上述したような板状体の場合には、該板状体の表面に試料を滴下したりすることにより接触させればよい。 When the non-absorbable carrier is a container as described above, for example, 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. Further, when 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.
　試料は、例えば希釈液により希釈して非吸収性担体に接触させることができる。試料の希釈液としては、トリス（ヒドロキシメチル）アミノメタン緩衝液、リン酸緩衝液、リン酸緩衝生理食塩水等の各種緩衝液または生理食塩水等を用いることができる。なお、上述した緩衝液のｐＨについては、４～１２の範囲内にあることが好ましい。 The sample can be diluted with, for example, a diluent and brought into contact with a non-absorbable carrier. As 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.
　また、試料の希釈液には、ウシ血清アルブミン、ヒト血清アルブミン、カゼインまたはその塩等の各種タンパク質、塩化ナトリウム等の各種塩類、各種糖類、脱脂粉乳、正常ウサギ血清等の各種動物血清、アジ化ナトリウム等の各種防腐剤、非イオン性界面活性剤、陽イオン性界面活性剤、陰イオン性界面活性剤、両性界面活性剤等の各種界面活性剤、ｎ-オクチル-β-Ｄ-グルコシド等の膜可溶化剤等の添加剤を適宜添加して用いることができる。 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-β-D-glucoside, etc. Additives such as membrane solubilizers can be appropriately added and used.
　これらの添加剤を加える際の濃度は特に限定されるものではないが、通常０．００１～１０％（ｗ／ｖｏｌ）、好ましくは０．０１～５％（ｗ／ｖｏｌ）である。 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).
　また、界面活性剤としては、例えばソルビタン脂肪酸エステル、グリセリン脂肪酸エステル、デカグリセリン脂肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エステル、ポリオキシエチレングリセリン脂肪酸エステル、ポリエチレングリコール脂肪酸エステル、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンフィトステロール、ポリオキシエチレンフィトスタノール、ポリオキシエチレンアルキルフェニルエーテル、ポリオキシエチレンヒマシ油、硬化ヒマシ油、ポリオキシエチレンラノリン等の非イオン性界面活性剤、例えばポリオキシエチレンアルキルエーテル酢酸塩、ポリオキシエチレンアルキルエーテル硫酸塩等の陰イオン性界面活性剤、例えば酢酸ベタイン等の両性界面活性剤等が挙げられる。また、膜可溶化剤としては、例えばｎ-オクチル-β-Ｄ-グルコシド、ｎ-オクチル-β-Ｄ-チオグルコシド、ｎ-ドデシル-β-Ｄ-マルトシド、ｎ-ノニル-β-Ｄ-チオマルトシド、ｎ-オクタノイル-Ｎ-メチル-Ｄ-グルカミン等が挙げられる。 Examples of the surfactant 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 Examples thereof include anionic surfactants such as alkyl ether sulfates, and amphoteric surfactants such as betaine acetate. Examples of membrane solubilizers include n-octyl-β-D-glucoside, n-octyl-β-D-thioglucoside, n-dodecyl-β-D-maltoside, and n-nonyl-β-D-thiomaltoside. N-octanoyl-N-methyl-D-glucamine and the like.
　非吸収性担体の表面に試料を接触させた後、この表面に接触させた試料を除去する。非吸収性担体の特異的結合物質が被覆されている面に接触させた試料の除去は、吸水性材料よりなる試料吸収体を非吸収性担体の表面上の試料に接触させて試料を吸収させたり、ピペット等で非吸収性担体の表面上の試料を吸い取ったり、または非吸収性担体を裏返して試料を下に落とすこと等により行うことができる。 ¡After bringing the sample into contact with the surface of the non-absorbable carrier, 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.
　なお、上述したように、試料吸収体やピペット等を用いて試料の除去を行う場合には、試料が、特異的結合物質が被覆されている部分の上を移動するように、特異的結合物質が被覆されている部分の水平方向から試料を吸収させたり、吸い取ることが好ましい。これにより、より高感度に測定対象物質を測定することができる。 As described above, when the sample is removed using a sample absorber, pipette, etc., 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.
　吸水性材料よりなる試料吸収体としては、例えばろ紙、ペーパータオル、ティッシュペーパー等の紙類、例えばポリエチレンもしくはポリスチレン等からなる焼結体、例えばポリビニルアルコールもしくはポリウレタン等からなるスポンジ、例えばレーヨンもしくはポリエステル等の化学繊維、布、不織布または綿等の液体を吸収する性質を持つ材料よりなるものを用いることができる。なお、試料吸収体の形状、大きさについては特に制限はない。このようにして試料を除去すると、試料が血液試料（全血試料）の場合、除去により血液試料中の赤血球は非吸収性担体の表面における特異的結合物質による被覆部分上より除去される。これに対して、血液試料中の測定対象物質は非吸収性担体の表面に固定化された特異的結合物質に結合して残る。そして、この結合した測定対象物質に特異的結合物質が固定化された粒子が結合するので、この結合した粒子の分布状態を赤血球に遮られずに明確に確認することができるのである。 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. A material made of a material having a property of absorbing liquid, such as chemical fiber, cloth, non-woven fabric, or cotton, can be used. In addition, there is no restriction | limiting in particular about the shape of a sample absorber, and a magnitude | size. 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 | grains by which the specific binding substance was fix | immobilized couple | bonded with this couple | bonded measuring object substance, the distribution state of this couple | bonded particle | grain can be confirmed clearly, without being interrupted by erythrocytes.
　なお、表面に接触させた試料を除去した後に、上述した試料の希釈液または緩衝液等で担体の表面を洗浄してもよい。 In addition, after removing the sample brought into contact with the surface, the surface of the carrier may be washed with the above-described sample dilution or buffer solution.
　表面に接触させた試料を除去した後には、測定対象物質に対する特異的結合物質であって上記非吸収性担体に固定化された特異的結合物質と同一または異なる特異的結合物質が固定化された粒子を、試料を除去した非吸収性担体の表面に接触させる。なお、試料中の複数の測定対象物質を測定する場合には、それぞれの測定対象物質に対する特異的結合物質を固定化した粒子を接触させる。 After removing the sample brought into contact with the surface, 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. When measuring a plurality of substances to be measured in a sample, particles on which a specific binding substance for each substance to be measured is immobilized are brought into contact with each other.
　また、特異的結合物質を固定化した粒子の代わりに、測定対象物質またはその類縁体を固定化した粒子を用いてもよい。ここで、測定対象物質の類縁体とは、測定対象物質の一部分、測定対象物質に別の物質が結合したもの、測定対象物質の構造の一部分が置換されたもの等であって、測定対象物質の特異的結合物質と結合する部分の構造を有し、特異的結合物質に結合することができる物質のことである。 Further, instead of 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. Here, 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.
　粒子は、例えば適当な分散媒に分散して非吸収性担体に接触させることができる。粒子の分散媒としては、トリス（ヒドロキシメチル）アミノメタン緩衝液、リン酸緩衝液、リン酸緩衝生理食塩水等の各種緩衝液または生理食塩水等を用いることができる。なお、上述した緩衝液のｐＨについては、４～１２の範囲内にあることが好ましい。 The particles can be dispersed, for example, in a suitable dispersion medium and brought into contact with a non-absorbable carrier. As the particle 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.
　また、粒子の分散媒には、上述した試料の希釈液の項で記載した添加剤をそれぞれ適宜添加して用いることができる。また、添加剤を加える際の濃度は特に限定されるものではないが、０．００１～１０％（ｗ／ｖｏｌ）が好ましく、なかでも、０．０１～５％（ｗ／ｖｏｌ）が好ましい。 In addition, the additives described in the section of the sample diluent described above can be appropriately added to the particle dispersion medium. The 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).
　図１は、担体として用いる平底マイクロプレート１であって、各ウェルの平底面２の片側半分３が特異的結合物質によって被覆されているものを示した図である。図１のＡは、平底マイクロプレート１の斜視図であり（斜線部３は、上記特異的結合物質で被覆されている部分である。）、図１のＢは、平底マイクロプレート１の平面図である（斜線部３は、上記特異的結合物質で被覆されている部分である。）。図１のＣは、平底マイクロプレート１を用い、かつ測定対象物質が存在する試料（「陽性」の試料）について測定を行った場合におけるウェルを上方から見た図である。図１のＤは、平底マイクロプレート１を用い、かつ測定対象物質が存在しない試料（「陰性」の試料）について測定を行った場合におけるウェルを上方から見た図である。なお、「陽性」の場合には、ウェルの平底面２の特異的結合物質によって被覆されていない部分から移動してきた粒子４は特異的結合物質被覆部分にトラップされる。なお、図中の矢印は、粒子４の移動方向を示す。 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), and 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. 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). In the case of “positive”, 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. In addition, the arrow in a figure shows the moving direction of the particle | grains 4. FIG.
　図２は、底面が長方形の直方体型透明容器５であって、特異的結合物質により帯状に被覆されている部分６（以下、帯状被覆部分と略記する場合がある。）を有する底面を有するものを示した図である。図２のＡは、該容器５の斜視図であり（斜線部６は、上記特異的結合物質で被覆されている部分である。）、図２のＢは、該容器５の平面図である（斜線部６は、上記特異的結合物質で被覆されている部分である。）。図２のＣは、底面が長方形の直方体型の透明容器５を用い、かつ測定対象物質が存在する試料（「陽性」の試料）について測定を行った場合における該容器５を上方から見た図である。図２のＤは、底面が長方形の直方体型の透明容器５を用い、かつ測定対象物質が存在しない試料（「陰性」の試料）について測定を行った場合における該容器５を上方から見た図である。なお、「陽性」の場合には、底面の特異的結合物質によって被覆されていない部分から移動してきた粒子４は帯状被覆部分６にトラップされる。なお、図中の矢印は、粒子４の移動方向を示す。 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. 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. In the case of “positive”, 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. In addition, the arrow in a figure shows the moving direction of the particle | grains 4. FIG.
　図３は、表面形状が長方形の板状体７であって、異なる２種類の特異的結合物質による帯状被覆部分８，８'を有する表面を有するものを示した図である。図３のＡは、該板状体７の斜視図であり（斜線部８，８'は、それぞれ異なる特異的結合物質で被覆されている部分である。）、図３のＢは、該板状体の平面図である（斜線部８，８'は、それぞれ異なる特異的結合物質で被覆されている部分である。）。図３のＣは、長方形の板状体７を用い、かつ２種類の測定対象物質が存在する試料（それぞれの測定対象物質が「陽性」の試料）について測定を行った場合における該板状体７を上方から見た図である。図３のＤは、長方形の板状体７を用い、かつ測定対象物質が存在しない試料（「陰性」の試料）について測定を行った場合における該板状体７を上方から見た図である。なお、「陽性」の場合には、表面の特異的結合物質によって被覆されていない部分から移動してきた粒子４はそれぞれの測定対象物質に対応する帯状被覆部分８，８'にトラップされる。なお、図中の矢印は、粒子４の移動方向を示す。 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. 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). . In the case of “positive”, 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. In addition, the arrow in a figure shows the moving direction of the particle | grains 4. FIG.
　図４は、表面形状が長方形の板状体９であって、該表面に断面形状が矩形の流路（溝）１０が設けられており、該流路（溝）１０の底面に異なる２種類の特異的結合物質による帯状被覆部分１１，１１'を形成したものを示した図である。図４のＡは、そのような板状体９の斜視図であり（斜線部１１，１１'は、それぞれ異なる特異的結合物質で被覆されている部分である。）、図４のＢは、そのような板状体９の平面図である（斜線部１１，１１'は、それぞれ異なる特異的結合物質で被覆されている部分である。）。また、流路（溝）１０の帯状被覆部分側に板１２を載せること等によりカバーすると（図４のＡ参照）、カバーされていない側の流路（溝）１０に滴下された試料は、毛細管現象で帯状被覆部分１１，１１'の方向に移動するので好ましい。図４のＣは、表面に流路（溝）１０が設けられた長方形の板状体９を用い、かつ２種類の測定対象物質が存在する試料（それぞれの測定対象物質が「陽性」の試料）について測定を行った場合における該板状体９を上方から見た図である。図４のＤは、表面に流路（溝）１０が設けられた長方形の板状体９を用い、かつ測定対象物質が存在しない試料（「陰性」の試料）について測定を行った場合における該板状体９を上方から見た図である。なお、「陽性」の場合には、流路（溝）１０の底面の特異的結合物質によって被覆されていない部分から移動してきた粒子４はそれぞれの測定対象物質に対応する帯状被覆部分１１，１１'にトラップされる。なお、図中の矢印は、粒子４の移動方向を示す。 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 | belt-shaped coating | coated parts 11 and 11 'with the specific binding substance of. 4A is a perspective view of such a plate-like body 9 (hatched portions 11 and 11 ′ are portions coated with different specific binding substances, respectively), and FIG. FIG. 2 is a plan view of such a plate-like body 9 (hatched portions 11, 11 ′ are portions coated with different specific binding substances). Further, when the plate 12 is covered by placing the plate 12 on the belt-like covering portion side of the channel (groove) 10 (see FIG. 4A), the sample dropped into the channel (groove) 10 on the uncovered side is It is preferable because it moves in the direction of the strip-shaped covering portions 11 and 11 ′ by capillary action. 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. In the case of “positive”, 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 '. In addition, the arrow in a figure shows the moving direction of the particle | grains 4. FIG.
　非吸収性担体の表面に粒子を接触させた後は、非吸収性担体の特異的結合物質により部分的に被覆されている表面における、特異的結合物質によって被覆されていない部分から被覆されている部分に粒子を移動させる。特異的結合物質によって被覆されていない部分から被覆されている部分に粒子を移動させる方法としては、例えば磁石を作用させる方法、非吸収性担体を傾ける方法等が挙げられる。 After the particles are brought into contact with the surface of the non-absorbable carrier, 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.
　磁石を作用させることによって粒子を移動させる場合には、粒子は、上述した磁性粒子を使用する。当該磁性粒子が、特異的結合物質により部分的に被覆されている表面における、特異的結合物質によって被覆されていない部分から被覆されている部分に移動するように磁石を作用させる。 When moving particles by applying a magnet, 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.
　非吸収性担体の表面上の磁性粒子の分布状態により陽性または陰性の判定を行なうことができる限り、非吸収性担体の表面に磁性粒子を接触させる前または接触させている間に磁石を作用させてもよい。 As long as a positive or negative determination can be made based on the distribution state of the magnetic particles on the surface of the non-absorbable carrier, 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.
　非吸収性担体の面が部分的に特異的結合物質によって被覆されている場合には、磁性粒子を移動させたい方向に移動させることができる位置であればどこに磁石を配置してもよく、例えば移動させたい方向やその周辺に配置すればよい。より具体的には、例えば表面の片側半分を特異的結合物質で被覆されている担体を用いた場合には、表面の特異的結合物質によって被覆されていない部分から被覆されている部分（図１の矢印方向）に磁性粒子が移動するように磁石を配置するのが好ましい。 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 | position to the direction to move, and its periphery. More specifically, for example, when a carrier in which one half of the surface is coated with a specific binding substance is used, a part coated from a part not covered with the specific binding substance on the surface (FIG. 1). It is preferable to arrange the magnets so that the magnetic particles move in the direction of the arrows.
　また、表面が特異的結合物質により帯状に被覆されている担体を用いた場合には、表面の特異的結合物質によって被覆されていない部分からその帯状被覆部分を通って特異的結合物質によって被覆されていない部分（図２～４の矢印方向）に磁性粒子が移動するように磁石を配置するのが好ましい。 In addition, when using a carrier whose surface is coated in a band with a specific binding substance, it is coated with a specific binding substance from the part not covered with the specific binding substance on the surface through the band-shaped coating part. It is preferable to arrange the magnets so that the magnetic particles move to the unexposed portions (arrow directions in FIGS. 2 to 4).
　磁石としては、磁場を発生して磁性粒子を磁化するものであればいずれのものでもよく、永久磁石、電磁石等を用いればよい。また、磁束密度は、用いる磁性粒子と担体表面との相互作用に依存するが、通常５～１００ガウスである。 As the magnet, 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.
　試料に測定対象物質が存在する場合には、該測定対象物質は非吸収性担体に固定化された特異的結合物質に結合する。この場合、磁性粒子に磁石を作用させると、該磁性粒子は磁石に吸引されて非吸収性担体の表面に沿って磁石の方向に移動するが、その過程で表面に固定化された特異的結合物質に結合した測定対象物質に出会うと、該磁性粒子は測定対象物質およびこれに結合している特異的結合物質を介して担体に結合して移動を停止するかまたは移動が著しく遅くなる。 When the substance to be measured exists in the sample, the substance to be measured binds to the specific binding substance immobilized on the non-absorbable carrier. In this case, when a magnet is allowed to act on the magnetic particles, the magnetic particles are attracted by the magnet and move in the direction of the magnet along the surface of the non-absorbent carrier. In the process, specific binding immobilized on the surface is performed. When the measurement target substance bound to the substance is encountered, the magnetic particles are bound to the carrier via the measurement target substance and the specific binding substance bound to the measurement target substance and stop moving, or the movement is significantly slowed down.
　担体表面の特異的結合物質が固定化されていない領域では、磁性粒子の移動は該粒子と担体表面との相互作用および磁場の強さに依存し、該磁性粒子は磁石の方向に速やかに移動する。一方、表面の特異的結合物質が固定化されている領域では、該表面に固定化されている特異的結合物質に、測定対象物質が結合している場合と結合していない場合とでは磁性粒子の移動速度に大きな差を生じる。 In the region where the specific binding substance on the carrier surface is not immobilized, 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. On the other hand, in the region where the specific binding substance on the surface is immobilized, 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.
　すなわち、試料中に測定対象物質が存在しない場合には、表面に固定化されている特異的結合物質は測定対象物質を結合しないので、磁性粒子は親和性を示さず、特異的結合物質が固定化されていない領域と同様に速やかに磁石の方向に移動する。従って、試料中に測定対象物質が存在しない場合には、磁性粒子は磁石に近い位置、すなわち、非吸収性担体の表面の端部に集まる。 That is, when there is no measurement target substance in the sample, 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.
　一方、試料中に測定対象物質が存在する場合には、表面に固定化されている特異的結合物質は測定対象物質を結合するので、磁性粒子は測定対象物質および特異的結合物質を介して非吸収性担体に結合して移動が停止または著しく遅くなる。従って、試料中に測定対象物質が存在する場合には、磁性粒子はその表面の特異的結合物質で被覆されている部分に集まる。 On the other hand, when the measurement target substance is present in the sample, 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.
　担体として容器を使用した場合、容器に添加された磁性粒子はその比重により容器の凹部の下方に沈降する。この際、容器の上部から底面方向に磁石を作用させることにより磁性粒子の沈降を促進してもよい。 When a container is used as the carrier, 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.
　非吸収性担体の特異的結合物質によって被覆されていない部分から被覆されている部分に移動するように磁性粒子に磁石を作用させる場合には、磁性粒子と測定対象物質が結合していない担体表面との相互作用は弱く、その移動速度はほぼ磁場の強さに依存する。従って、大きな移動速度を必要とする場合、すなわち、短時間で測定結果を求める時には強い磁場を発生する磁石を使用すればよい。また、電磁石を用いて磁場の強さを調節しながら測定を行うことも可能である。 When a magnet is allowed to act on the magnetic particles so that they move from a portion that is not coated with a specific binding substance of the non-absorbable carrier to a portion that is coated, the surface of the carrier on which the magnetic particles and the substance to be measured are not bound Interaction is weak, and its moving speed depends almost on the strength of the magnetic field. Therefore, when a large moving speed is required, that is, when a measurement result is obtained in a short time, a magnet that generates a strong magnetic field may be used. It is also possible to perform measurement while adjusting the strength of the magnetic field using an electromagnet.
　非吸収性担体を傾けることにより移動させる場合には、粒子が非吸収性担体の特異的結合物質により部分的に被覆されている表面における、特異的結合物質によって被覆されていない部分から被覆されている部分に移動するように非吸収性担体を傾けて、重力により粒子を移動させる。なお、非吸収性担体を傾ける角度は、粒子が重力によって非吸収性担体の特異的結合物質により部分的に被覆されている表面における、特異的結合物質によって被覆されていない部分から被覆されている部分に移動するような角度であればよく、９０°以下の角度を適宜選択すればよいが、２５°から９０°の間の角度が好ましく、なかでも、４５°から６５°の間の角度が好ましい。 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.
　非吸収性担体の表面上の粒子の分布状態により陽性または陰性の判定を行なうことができる限り、担体表面に粒子を接触させる前または接触させている間に担体を傾けてもよい。 As long as positive or negative determination can be made based on the distribution state of the particles on the surface of the non-absorbable carrier, the carrier may be tilted before or while the particles are brought into contact with the carrier surface.
　重力により粒子を移動させる場合には、上述したように、例えば移動させたい方向を下にして非吸収性担体を配置すればよく、より具体的には、例えば表面の片側半分を特異的結合物質で被覆されている非吸収性担体を用いた場合には、表面の特異的結合物質によって被覆されていない部分から被覆されている部分（図１の矢印方向）に粒子が移動するように、非吸収性担体を傾けるのが好ましい。 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. In the case of using a non-absorbable carrier coated with a non-absorbable carrier, 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.
　また、表面が特異的結合物質により帯状に被覆されている担体を用いた場合には、表面の特異的結合物質によって被覆されていない部分からその帯状被覆部分を通って特異的結合物質によって被覆されていない部分（図２～４の矢印方向）に粒子が移動するように担体を傾けるのが好ましい。 In addition, when using a carrier whose surface is coated in a band with a specific binding substance, it is coated with a specific binding substance from the part not covered with the specific binding substance on the surface through the band-shaped coating part. It is preferable to incline the carrier so that the particles move to the unexposed portion (arrow direction in FIGS. 2 to 4).
　試料に測定対象物質が存在する場合には、該測定対象物質は非吸収性担体に固定化された特異的結合物質に結合する。この場合、粒子が非吸収性担体の特異的結合物質で被覆されている面に沿って移動するように非吸収性担体を傾けると、該粒子は重力により非吸収性担体の表面に沿って傾きの下方向に移動するが、その過程で表面に固定化された特異的結合物質に結合した測定対象物質に出会うと、該粒子は測定対象物質およびこれに結合している特異的結合物質を介して担体に結合して移動を停止するかまたは移動が著しく遅くなる。 When the substance to be measured exists in the sample, the substance to be measured binds to the specific binding substance immobilized on the non-absorbable carrier. In this case, when 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. When 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.
　担体表面の特異的結合物質が固定化されていない領域では、粒子の移動は該粒子と担体表面との相互作用および担体を傾ける角度に依存し、該粒子は傾きの下方向に速やかに移動する。一方、表面の特異的結合物質が固定化されている領域では、該表面に固定化されている特異的結合物質に、測定対象物質が結合している場合と結合していない場合とでは粒子の移動速度に大きな差を生じる。 In the region where the specific binding substance on the carrier surface is not immobilized, 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. . On the other hand, in the region where the specific binding substance on the surface is immobilized, 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.
　すなわち、試料中に測定対象物質が存在しない場合には、表面に固定化されている特異的結合物質は測定対象物質を結合しないので、粒子は親和性を示さず、特異的結合物質が固定化されていない領域と同様に速やかに傾きの下方向に移動する。従って、試料中に測定対象物質が存在しない場合には、粒子は担体の下方向の端部に集まる。 That is, when there is no measurement target substance in the sample, 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.
　一方、試料中に測定対象物質が存在する場合には、表面に固定化されている特異的結合物質は測定対象物質を結合するので、粒子は測定対象物質および特異的結合物質を介して非吸収性担体に結合して移動が停止または著しく遅くなる。従って、試料中に測定対象物質が存在する場合には、粒子はその表面の特異的結合物質で被覆されている部分に集まる。 On the other hand, when the measurement target substance exists in the sample, 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.
　非吸収性担体を傾けて粒子を非吸収性担体の特異的結合物質によって被覆されていない部分から被覆されている部分に移動させる場合には、粒子と測定対象物質が結合していない担体表面との相互作用は弱く、その移動速度は非吸収性担体を傾ける角度にほぼ依存する。従って、大きな移動速度を必要とする場合、すなわち、短時間で測定結果を求める時には非吸収性担体を傾ける角度を大きくすればよい。また、非吸収性担体を傾ける角度を経時的に変化させてもよい。さらに、板状体の非吸収性担体を用いる場合には、その板状体を湾曲させ板状体の角度を変化させることにより、粒子が移動する速度を変化させてもよい。 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 | carrier over time. Further, when a non-absorbable carrier having a plate-like body is used, the moving speed of the particles may be changed by curving the plate-like body and changing the angle of the plate-like body.
　本発明において、非吸収性担体の表面に特異的結合物質を固定化する場合には、該表面が部分的に該特異的結合物質で被覆されるように、特異的結合物質を塗布・固定化することが好ましい。これは、試料中に測定対象物質が存在する場合において、特異的結合物質で被覆されている部分の像と被覆されていない部分の像を比較することによって、「陽性」であることの判定がより明確となり、「陽性」と「陰性」の判別、すなわち、試料中の測定対象物質の有無の判別が容易になる。ここで、「部分的」とは、特異的結合物質が偏在しており、表面全体にわたって全面的に特異的結合物質で被覆されていないことをいう。特異的結合物質で被覆されている部分の形状および面積は、試料中の測定対象物質の存在の有無を判別することができる限り特に制限はない。部分的被覆の例としては、担体表面の片側半分を被覆したり、非吸収性担体の表面を帯状、文字、図形等の各種形状に被覆する例が挙げられる。 In the present invention, 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. Here, “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.
　また、非吸収性担体の表面の特異的結合物質による被覆部分は複数存在していてもよく、例えば試料中の複数の測定対象物質を測定するために、非吸収性担体の表面にそれぞれの測定対象物質に対する特異的結合物質による被覆部分を設けることができる。 In addition, there may be a plurality of coating portions of the surface of the non-absorbable carrier with specific binding substances. For example, in order to measure a plurality of substances to be measured in a sample, each surface of the non-absorbable carrier is measured. A coating portion with a specific binding substance for the target substance can be provided.
　上記のように、粒子を非吸収性担体の特異的結合物質によって被覆されていない部分から被覆されている部分に移動させた後、表面における粒子の分布状態から測定対象物質の有無を判定する。非吸収性担体の表面上の粒子の分布状態、言い換えれば、粒子の分布像は、肉眼、または吸光度測定やパターン認識によるマイクロプレートリーダ等の光学的読み取り装置により確認することができる。 As described above, after the particles are moved from the portion that is not coated with the specific binding substance of the non-absorbable carrier to the portion that is coated, 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.
　なお、特異的結合物質の代わりに、測定対象物質またはその類縁体を固定化した粒子を用いた場合には、上述した測定方法により得られる「陽性」または「陰性」の粒子の分布状態と逆の結果が得られる。 In addition, in the case where particles to which the measurement target substance or its analog is immobilized are used instead of the specific binding substance, the distribution state of the “positive” or “negative” particles obtained by the measurement method described above is reversed. Result is obtained.
　本発明の非吸収性担体を用いた測定方法の別法としては、例えば上述したように特異的結合物質を固定化し、該特異的結合物質で部分的に被覆されている面、すなわち、特異的結合物質によって被覆されていない部分と被覆されている部分を併せ持つ面を有する非吸収性担体の表面に、特異的結合物質が固定化された粒子および試料を混合した混合物を非吸収性担体の表面に接触させる方法が挙げられる（以下、本発明の第２の測定方法と略記する場合がある。）。 As another method of the measurement method using the non-absorbable carrier of the present invention, for example, as described above, 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.).
　非吸収性担体の表面に、特異的結合物質が固定化された粒子および試料を混合した混合物を接触させた後、該混合物を担体表面の特異的結合物質で被覆されている部分の上を移動させ、担体表面における粒子の分布状態から測定対象物質の有無を判定する。 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.
　上述した第２の測定方法によれば、試料中に測定対象物質が存在する、すなわち、「陽性」の場合においては、粒子は試料中の測定対象物質と結合するので、粒子および試料の混合物を非吸収性担体の表面に接触させた時に、粒子は測定対象物質を介して担体表面に固定化された特異的結合物質と結合する。次いで、粒子および試料の混合物を担体表面から除去し、さらに粒子を担体表面に接触させて面に沿って移動させると、該粒子は先に非吸収性担体の面に測定対象物質を介して結合した粒子に遮られて移動が停止または著しく遅くなり、粒子は担体表面の特異的結合物質で被覆されている部分に集まる。 According to the second measurement method described above, when the measurement target substance exists in the sample, that is, in the case of “positive”, the particles bind to the measurement target substance in the sample. 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. Next, when the mixture of the particles and the sample is removed from the carrier surface, and further the particles are brought into contact with the carrier surface and moved along the surface, 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.
　一方、試料中に測定対象物質が存在しない、すなわち、「陰性」の場合においては、粒子は測定対象物質を結合しないので、粒子および試料の混合物を非吸収性担体の面に接触させた時に、粒子は担体表面にあらかじめ固定化されていた特異的結合物質とは結合しない。よって、粒子および試料の混合物を担体表面から除去し、さらに粒子を担体表面に接触させて面に沿って移動させても、粒子は担体表面の上を素通りしてしまい速やかに磁石の方向または傾きの下方向に移動して、磁石に近い位置、すなわち、担体の面の端部、あるいは担体の下方向の担部に集まる。従って、このような測定方法によれば、第１の測定方法により得られる「陽性」または「陰性」の粒子の分布状態と同じ結果が得られる。 On the other hand, when the measurement target substance does not exist in the sample, that is, in the case of “negative”, 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.
　なお、本発明の測定方法は、確認試験に使用することができる。確認試験とは、測定によって「陽性」の像が得られた場合に、その像が、真に測定対象物質の存在によるものか、あるいは非特異的凝集反応によるものかを確認する方法である。 Note that 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.
　また、粒子として、蛍光色素で着色したものを使用する場合における標識方法や検出方法については、例えば特開２０１０－１４６３１号公報、特開２０１３－２００３１６号公報、特開２０１３－２２８３０８号公報等に準じて行えばよい。 For the labeling method and detection method when particles colored with a fluorescent dye are used, for example, in JP 2010-14631 A, JP 2013-200316 A, JP 2013-228308 A, etc. It may be done according to this.
　以下、実施例および比較例に基づいて本発明を具体的に説明するが、本発明はこれらの例によって何ら限定されるものではない。なお、特記しない限り、％は、重量／体積基準（ｗ／ｖｏｌ％）である。 Hereinafter, the present invention will be specifically described based on Examples and Comparative Examples, but the present invention is not limited to these examples. Unless otherwise specified,% is based on weight / volume (w / vol%).
　実施例１　抗体を塗布した本発明に係るプラスチック基板の作製（本発明の非吸収性担体の作製）
　ポリスチレン基板１６（幅１０ｍｍ、奥行き３３ｍｍ、厚さ１ｍｍ）上の両端に２枚のポリスチレン板１４，１４'（幅２ｍｍ、奥行き３３ｍｍ、厚さ０．５ｍｍ）をそれぞれ貼り合わせて、図５に示される、表面に流路（溝）１０（幅６ｍｍ、奥行き３３ｍｍ、高さ０．５ｍｍ）が設けられた板状体１３を作製した。１０ｍＭのリン酸緩衝生理食塩水（ＰＢＳ；ｐＨ７．０）および０．０３％のｎ-オクチル-β-Ｄ-チオグルコシド水溶液を用い、抗Ａ型インフルエンザモノクローナル抗体または抗Ｂ型インフルエンザモノクローナル抗体を、該抗体の抗体濃度が１ｍｇ／ｍＬになるように希釈して抗体溶液とした。パルスインジェクター塗布装置（クラスターテクノロジー（株）社製）を用いて、上記ポリスチレン板状体１３の流路（溝）１０の底面の手前から１４ｍｍの位置に、上記抗体溶液を約１ｍｍのライン状に塗布した。抗体を塗布したポリスチレン板状体１３を、ブロッキング液（５０ｍＭのトリス塩酸緩衝液（ＴＢＳ；ｐＨ７．０）、０．５％のカゼイン、５％の乳糖、０．０９％のアジ化ナトリウム）に６℃で１晩浸漬し、ブロッキング処理を行った。次いで、ブロッキング処理したポリスチレン板状体１３の水分を除去した後、減圧下で１晩静置して乾燥させた。乾燥したポリスチレン板状体１３に対し、一方にポリオレフィン系繊維からなる吸収体、他方に滴下口を有したカバー１２を取り付け、テストデバイスとした。 Example 1 Production of a plastic substrate according to the present invention coated with an antibody (production of a non-absorbable carrier of the present invention)
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. Using 10 mM phosphate buffered saline (PBS; pH 7.0) and 0.03% aqueous n-octyl-β-D-thioglucoside, anti-influenza A monoclonal antibody or anti-influenza B monoclonal antibody, An antibody solution was prepared by diluting the antibody so that the antibody concentration was 1 mg / mL. Using 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.
　実験例１　インフルエンザウイルスの測定
　（１）抗体を感作させた磁性粒子液の作製方法
　ダイナビーズ（登録商標）Ｍ-４５０ Epoxy（ライフテクノロジーズ社製）４．６ｍＬを量り取り、磁石を押し当てて磁性粒子を集めた後、上清をアスピレーターで吸引し、２．３ｍＬの精製水で洗浄した。該磁性粒子を感作緩衝液（１０ｍＭのＰＢ、１ＭＮａＣｌ、ｐＨ７．２）２．１ｍＬに懸濁し、該感作緩衝液に抗Ａ型インフルエンザモノクローナル抗体（３．２ｍｇ／ｍＬ）０．１ｍＬと抗Ｂ型インフルエンザモノクローナル抗体（４．５ｍｇ／ｍＬ）０．１ｍＬを添加した。次いで、得られた溶液を回転振とうさせながら、３７℃で２時間感作を行った後、ブロッキング液（５０ｍＭのトリス塩酸緩衝液（ＴＢＳ；ｐＨ８．０）、０．５％のカゼイン、０．０９％のアジ化ナトリウム）６．９ｍＬを添加し、回転振とうさせながら、３７℃で１時間ブロッキング処理を行った。ブロッキング処理した後の磁性粒子に磁石を押し当て磁性粒子を集めた後、ブロッキング液の上清をアスピレーターで吸引した。該磁性粒子に、再度ブロッキング液３３ｍＬを添加して磁性粒子をよく懸濁し、磁石を押し当て磁性粒子を集めた後、ブロッキング液の上清をアスピレーターで吸引する洗浄処理を合計５回行った。洗浄処理した後の磁性粒子に、分散緩衝液（５０ｍＭのトリス塩酸緩衝液（ＴＢＳ；ｐＨ８．８）、０．５％のカゼイン、１０％の非動化済みマウス血清、０．０９％アジ化ナトリウム）７０ｍＬを添加しよく懸濁させて、抗体を感作させた磁性粒子液とした。 Experimental Example 1 Measurement of Influenza Virus (1) Preparation Method of Magnetic Particle Solution Sensitized with Antibody Dynabes (registered trademark) M-450 Epoxy (manufactured by Life Technologies) 4.6mL was weighed and magnet was pressed After collecting the magnetic particles, the supernatant was aspirated and washed with 2.3 mL of purified water. The magnetic particles are suspended in 2.1 mL of sensitization buffer (10 mM PB, 1M NaCl, pH 7.2), and 0.1 mL of anti-influenza A monoclonal antibody (3.2 mg / mL) and anti-antigen are added to the sensitization buffer. 0.1 mL of influenza B monoclonal antibody (4.5 mg / mL) was added. Next, 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. 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. 33 mL of blocking solution was again added to the magnetic particles to sufficiently suspend the magnetic particles, the magnets were pressed to collect the magnetic particles, and then the washing treatment of sucking the supernatant of the blocking solution with an aspirator was performed a total of 5 times. The magnetic particles after the washing treatment were subjected to dispersion buffer (50 mM Tris-HCl buffer (TBS; pH 8.8), 0.5% casein, 10% non-immobilized mouse serum, 0.09% azide). Sodium) (70 mL) was added and well suspended to obtain a magnetic particle solution sensitized with an antibody.
　（２）インフルエンザウイルスの測定方法
　Ａ型インフルエンザウイルス培養液（４．０×１０７ｐｆｕ／ｍＬ）またはＢ型インフルエンザウイルス培養液（２．２×１０７ｐｆｕ／ｍＬ）を、０．５％のカゼインを含む５０ｍＭのトリス塩酸緩衝液（ＴＢＳ；ｐＨ８．０）で希釈し、４０倍希釈、８０倍希釈、１６０倍希釈、３２０倍希釈および６４０倍希釈の試料を作製した。次いで、実施例１のテストデバイスをＰＵＬＬＵＮＯ（和光純薬工業（株）製）のスライド板にセットし、上記５種類の試料を検体希釈液（界面活性剤を含む５０ｍＭのトリス塩酸緩衝液（ｐＨ８．０）、０．５ＭのＮａＣｌ、０．５％のカゼイン、０．００１５％のブリリアントブルー、０．０９％のアジ化ナトリウム）で１０倍希釈した希釈試料６０μＬをそれぞれテストデバイスの適下口に滴下し、希釈液が測定レーン上を流れて吸収体に全て吸収されたことを目視で確認した後、上記の磁性粒子液５０μＬを滴下口に滴下した。ＰＵＬＬＵＮＯのスライド板をマグネット側にいっぱいまで押して測定を開始し、１分後にスライド板が元の位置に戻った時点で、「陽性」／「陰性」の判定を行った。なお、ブランク測定は、６０μＬの検体希釈液のみをテストデバイスに滴下し、上記と同様の方法で１０回測定を行い、それぞれについて、「陽性」／「陰性」の判定を行った。判定の結果を表１および表２に示す。なお、表１および表２において、判定部に赤褐色のラインが確認できた場合を「＋」、薄い赤褐色のラインが確認できた場合を「＋ｗ」とし、これらはいずれも「陽性」と判定した。また、判例部に赤褐色のラインが確認できなかった場合を「－」と判定した。 (2) 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. Next, 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 μL 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 μL 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. When the slide plate returned to its original position after 1 minute, “Positive” / “Negative” determination was performed. In the blank measurement, only 60 μL of the specimen dilution solution was dropped on the test device, the measurement was performed 10 times in the same manner as described above, and “positive” / “negative” was determined for each. The determination results are shown in Tables 1 and 2. In Tables 1 and 2, the case where a reddish brown line was confirmed in the determination part was “+”, and the case where a light reddish brown line was confirmed was “+ w”, both of which were determined as “positive”. . Further, when a reddish brown line could not be confirmed in the case part, it was determined as “−”.
　比較例１　抗体を塗布した比較用のプラスチック基板Ａの作製（比較用の非吸収性担体Ａの作製）
　ポリスチレン基板１６（幅１０ｍｍ、奥行き３３ｍｍ、厚さ１ｍｍ）上の両端に２枚のポリスチレン板１４，１４'（幅２ｍｍ、奥行き３３ｍｍ、厚さ０．５ｍｍ）をそれぞれ貼り合わせて、図５に示される、表面に流路（溝）１０（幅６ｍｍ、奥行き３３ｍｍ、高さ０．５ｍｍ）が設けられた板状体１３を作製した。１０ｍＭのリン酸緩衝生理食塩水（ＰＢＳ；ｐＨ７．０）を用い、抗Ａ型インフルエンザモノクローナル抗体または抗Ｂ型インフルエンザモノクローナル抗体を、該抗体の抗体濃度が１ｍｇ／ｍＬになるように希釈して抗体溶液とした。パルスインジェクター塗布装置（クラスターテクノロジー（株）社製）を用いて、上記ポリスチレン板状体１３の流路（溝）１０の底面の手前から１４ｍｍの位置に、上記抗体溶液を約１ｍｍのライン状に塗布した。抗体を塗布したポリスチレン板状体１３を、ブロッキング液（５０ｍＭのトリス塩酸緩衝液（ＴＢＳ；ｐＨ７．０）、０．５％のカゼイン、５％の乳糖、０．０９％のアジ化ナトリウム）と０．０３％のｎ-オクチル-β-Ｄ-チオグルコシド水溶液の混合液に６℃で１晩浸漬し、ブロッキング処理を行った。次いで、ブロッキング処理したポリスチレン板状体１３の水分を除去した後、減圧下で１晩静置して乾燥させた。乾燥したポリスチレン板状体１３に対し、一方にポリオレフィン系繊維からなる吸収体、他方に滴下口を有したカバー１２を取り付け、テストデバイスとした。 Comparative Example 1 Preparation of comparative plastic substrate A coated with antibody (Preparation of non-absorbable carrier A for comparison)
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. Using 10 mM phosphate buffered saline (PBS; pH 7.0), 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. Using 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 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-β-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.
　実験例２　インフルエンザウイルスの測定
　比較例１のテストデバイスを用いた以外は、実験例１と同様の操作で、インフルエンザウイルスの測定を行った。判定の結果を表１および表２に示す。 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.
　比較例２　抗体を塗布した比較用のプラスチック基板Ｂの作製（比較用の非吸収性担体Ｂの作製）
　ポリスチレン基板１６（幅１０ｍｍ、奥行き３３ｍｍ、厚さ１ｍｍ）上の両端に２枚のポリスチレン板１４，１４'（幅２ｍｍ、奥行き３３ｍｍ、厚さ０．５ｍｍ）をそれぞれ貼り合わせて、図５に示される、表面に流路（溝）１０（幅６ｍｍ、奥行き３３ｍｍ、高さ０．５ｍｍ）が設けられた板状体１３を作製した。１０ｍＭのリン酸緩衝生理食塩水（ＰＢＳ；ｐＨ７．０）を用い、抗Ａ型インフルエンザモノクローナル抗体または抗Ｂ型インフルエンザモノクローナル抗体を、該抗体の抗体濃度が１ｍｇ／ｍＬになるように希釈して抗体溶液とした。一方、０．０３％のｎ-オクチル-β-Ｄ-チオグルコシド水溶液に、ポリスチレン板状体１３を２５℃で２時間浸漬し、該ポリスチレン板状体の表面にｎ-オクチル-β-Ｄ-チオグルコシドを被覆した。次いで、該ポリスチレン板状体１３の水分を除去した後、減圧下で２時間静置して乾燥させた。さらに、パルスインジェクター塗布装置（クラスターテクノロジー（株）社製）を用いて、上記ポリスチレン板状体１３の流路（溝）１０の底面の手前から１４ｍｍの位置に、上記抗体溶液を約１ｍｍのライン状に塗布した。抗体を塗布したポリスチレン板状体１３を、ブロッキング液（５０ｍＭのトリス塩酸緩衝液（ＴＢＳ；ｐＨ７．０）、０．５％のカゼイン、５％の乳糖、０．０９％のアジ化ナトリウム）に６℃で１晩浸漬し、ブロッキング処理を行った。次いで、ブロッキング処理したポリスチレン板状体１３の水分を除去した後、減圧下で１晩静置して乾燥させた。乾燥したポリスチレン板状体１３に対し、一方にポリオレフィン系繊維からなる吸収体、他方に滴下口を有したカバー１２を取り付け、テストデバイスとした。 Comparative Example 2 Production of Comparative Plastic Substrate B Coated with Antibody (Production of Non-absorbable Carrier B for Comparison)
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. Using 10 mM phosphate buffered saline (PBS; pH 7.0), 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. On the other hand, the polystyrene plate 13 was immersed in a 0.03% aqueous solution of n-octyl-β-D-thioglucoside at 25 ° C. for 2 hours, and the surface of the polystyrene plate was n-octyl-β-D-. Coated with thioglucoside. Subsequently, after removing the water | moisture content of this polystyrene plate-shaped object 13, it left still under reduced pressure for 2 hours and was dried. Furthermore, using a pulse injector coating device (manufactured by Cluster Technology Co., Ltd.), 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. 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.
　実験例３　インフルエンザウイルスの測定
　比較例２のテストデバイスを用いた以外は、実験例１と同様の操作で、インフルエンザウイルスの測定を行った。判定の結果を表１および表２に示す。 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.
　比較例３　抗体を塗布した比較用のプラスチック基板Ｃの作製（比較用の非吸収性担体Ｃの作製）
　ポリスチレン基板１６（幅１０ｍｍ、奥行き３３ｍｍ、厚さ１ｍｍ）上の両端に２枚のポリスチレン板１４，１４'（幅２ｍｍ、奥行き３３ｍｍ、厚さ０．５ｍｍ）をそれぞれ貼り合わせて、図５に示される、表面に流路（溝）１０（幅６ｍｍ、奥行き３３ｍｍ、高さ０．５ｍｍ）が設けられた板状体１３を作製した。１０ｍＭのリン酸緩衝生理食塩水（ＰＢＳ；ｐＨ７．０）を用い、抗Ａ型インフルエンザモノクローナル抗体または抗Ｂ型インフルエンザモノクローナル抗体を、該抗体の抗体濃度が１ｍｇ／ｍＬになるように希釈して抗体溶液とした。パルスインジェクター塗布装置（クラスターテクノロジー（株）社製）を用いて、上記ポリスチレン板状体１３の流路（溝）１０の底面の手前から１４ｍｍの位置に、上記抗体溶液をライン状に塗布した。抗体を塗布したポリスチレン板状体１３を、０．０３％のｎ-オクチル-β-Ｄ-チオグルコシド水溶液に２５℃で２時間浸漬し、ポリスチレン板状体１３の表面にｎ-オクチル-β-Ｄ-チオグルコシドを被覆した。次いで、ポリスチレン板状体１３の水分を除去した後、減圧下で２時間静置して乾燥させた。乾燥させたポリスチレン板状体１３は、ブロッキング液（５０ｍＭのトリス塩酸緩衝液（ＴＢＳ；ｐＨ７．０）、０．５％のカゼイン、５％の乳糖、０．０９％のアジ化ナトリウム）に６℃で１晩浸漬し、ブロッキング処理を行った。次いで、ブロッキング処理したポリスチレン板状体１３の水分を除去した後、減圧下で１晩静置して乾燥させた。乾燥したポリスチレン板状体１３に対し、一方にポリオレフィン系繊維からなる吸収体、他方に滴下口を有したカバー１２を取り付け、テストデバイスとした。 Comparative Example 3 Production of Comparative Plastic Substrate C Coated with Antibody (Production of Non-absorbable Carrier C for Comparison)
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. Using 10 mM phosphate buffered saline (PBS; pH 7.0), 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-β-D-thioglucoside at 25 ° C. for 2 hours, and the surface of the polystyrene plate 13 was n-octyl-β-. D-thioglucoside was coated. Subsequently, after removing the water | moisture content of the polystyrene plate-shaped body 13, it left still under reduced pressure for 2 hours and was made to dry. The dried polystyrene plate 13 was added 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 overnight at 0 ° C. 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.
　実験例４　インフルエンザウイルスの測定
　比較例３のテストデバイスを用いた以外は、実験例１と同様の操作で、インフルエンザウイルスの測定を行った。判定の結果を表１および表２に示す。 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.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　実験例１～４の結果から明らかなように、比較用のプラスチック基板Ａ～Ｃを用いた場合には、ブランク測定において「陽性」反応が発現してしまい、特異的かつ確度の高い測定ができないことがわかった。その一方で、本発明の固定化方法によって抗体を固定化させたポリスチレン板状体（本発明の非吸収性担体）を用いた場合には、ブランク測定において「陽性」反応が発現することなく、すなわち、非特異反応を抑制しつつ、インフルエンザウイルスを特異的かつ精度よく測定できることがわかった。これらの結果から、例えばｎ-オクチル-β-Ｄ-チオグルコシド等の膜可溶化剤を、抗原等の特異的結合物質と混合させ、その混合液を非吸収性担体に塗布することによってのみ、非特異反応を抑制できることがわかった。 As is clear from the results of Experimental Examples 1 to 4, when the plastic substrates A to C for comparison are used, a “positive” reaction appears in the blank measurement, and a specific and highly accurate measurement cannot be performed. I understood it. On the other hand, when using a polystyrene plate (non-absorbable carrier of the present invention) on which an antibody is immobilized by the immobilization method of the present invention, a “positive” reaction is not expressed in the blank measurement, That is, it was found that influenza virus can be measured specifically and accurately while suppressing non-specific reactions. From these results, for example, only by mixing a membrane solubilizer such as n-octyl-β-D-thioglucoside with a specific binding substance such as an antigen, and applying the mixture to a non-absorbable carrier, It was found that non-specific reactions can be suppressed.
　実施例２～６および比較例４～６　各種膜可溶化剤を用いたプラスチック基板の作製
　ポリスチレン基板１６（幅１０ｍｍ、奥行き３３ｍｍ、厚さ１ｍｍ）上の両端に２枚のポリスチレン板１４，１４'（幅２ｍｍ、奥行き３３ｍｍ、厚さ０．５ｍｍ）をそれぞれ貼り合わせて、図５に示される、表面に流路（溝）１０（幅６ｍｍ、奥行き３３ｍｍ、高さ０．５ｍｍ）が設けられた板状体１３を作製した。１０ｍＭのリン酸緩衝生理食塩水（ＰＢＳ；ｐＨ７．０）および表３に示す各種膜可溶化剤を用い、かつ該膜可溶化剤の濃度が表３に示す特定濃度になるように膜可溶化剤を希釈した水溶液を用い、抗Ａ型インフルエンザモノクローナル抗体を、該抗体の抗体濃度が０．２ｍｇ／ｍＬになるように希釈して抗体溶液とした。パルスインジェクター塗布装置（クラスターテクノロジー（株）社製）を用いて、上記ポリスチレン板状体１３の流路（溝）１０の底面の手前から１４ｍｍの位置に、上記抗体溶液を約１ｍｍのライン状に塗布した。抗体を塗布したポリスチレン板状体１３を、ブロッキング液（５０ｍＭのトリス塩酸緩衝液（ＴＢＳ；ｐＨ７．０）、０．５％のカゼイン、５％の乳糖、０．０９％のアジ化ナトリウム）に６℃で１晩浸漬し、ブロッキング処理を行った。次いで、ブロッキング処理したポリスチレン板状体１３の水分を除去した後、減圧下で１晩静置して乾燥させた。乾燥したポリスチレン板状体１３に対し、一方にポリオレフィン系繊維からなる吸収体、他方に滴下口を有したカバー１２を取り付け、テストデバイスとした。 Examples 2 to 6 and Comparative Examples 4 to 6 Production of Plastic Substrates Using Various Membrane Solubilizing Agents Two polystyrene plates 14, 14 ′ at both ends on a polystyrene substrate 16 (width 10 mm, depth 33 mm, thickness 1 mm) (Width 2 mm, depth 33 mm, thickness 0.5 mm) were bonded to each other, and a flow path (groove) 10 (width 6 mm, depth 33 mm, height 0.5 mm) was provided on the surface shown in FIG. A plate-like body 13 was produced. Using 10 mM phosphate buffered saline (PBS; pH 7.0) and various membrane solubilizers shown in Table 3, solubilizing the membrane so that the concentration of the membrane solubilizer becomes the specific concentration shown in Table 3 Using an aqueous solution in which the agent was diluted, 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. Using 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.
　実験例５～１２　インフルエンザウイルスの測定
　実施例２～６および比較例４～６のテストデバイスを用いた以外は、実験例１と同様の操作で、インフルエンザウイルスの測定を行った。判定の結果を表３に示す。 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.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　比較例７～１２　各種添加剤を用いたプラスチック基板の作製
　ポリスチレン基板１６（幅１０ｍｍ、奥行き３３ｍｍ、厚さ１ｍｍ）上の両端に２枚のポリスチレン板１４，１４'（幅２ｍｍ、奥行き３３ｍｍ、厚さ０．５ｍｍ）をそれぞれ貼り合わせて、図５に示される、表面に流路（溝）１０（幅６ｍｍ、奥行き３３ｍｍ、高さ０．５ｍｍ）が設けられた板状体１３を作製した。１０ｍＭのリン酸緩衝生理食塩水（ＰＢＳ；ｐＨ７．０）および表４に示す各種添加剤を用い、かつ該添加剤の濃度が表４に示す特定濃度になるように添加剤を希釈した水溶液を用い、抗Ａ型インフルエンザモノクローナル抗体を、該抗体の抗体濃度が０．２ｍｇ／ｍＬになるように希釈して抗体溶液とした。パルスインジェクター塗布装置（クラスターテクノロジー（株）社製）を用いて、上記ポリスチレン板状体１３の流路（溝）１０の底面の手前から１４ｍｍの位置に、上記抗体溶液を約１ｍｍのライン状に塗布した。抗体を塗布したポリスチレン板状体１３を、ブロッキング液（５０ｍＭのトリス塩酸緩衝液（ＴＢＳ；ｐＨ７．０）、０．５％のカゼイン、５％の乳糖、０．０９％のアジ化ナトリウム）に６℃で１晩浸漬し、ブロッキング処理を行った。次いで、ブロッキング処理したポリスチレン板状体１３の水分を除去した後、減圧下で１晩静置して乾燥させた。乾燥したポリスチレン板状体１３に対し、一方にポリオレフィン系繊維からなる吸収体、他方に滴下口を有したカバー１２を取り付け、テストデバイスとした。 Comparative Examples 7 to 12 Production of plastic substrate using various additives Two polystyrene plates 14, 14 '(width 2mm, depth 33mm, thickness) on both ends of polystyrene substrate 16 (width 10mm, depth 33mm, thickness 1mm) The plate-like body 13 provided with a flow path (groove) 10 (width 6 mm, depth 33 mm, height 0.5 mm) on the surface as shown in FIG. 5 was produced. An aqueous solution in which 10 mM phosphate buffered saline (PBS; pH 7.0) and various additives shown in Table 4 were used, and the additives were diluted so that the concentration of the additive became a specific concentration shown in Table 4 was prepared. 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. Using 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.
　実験例１３～１８　インフルエンザウイルスの測定
　比較例７～１２のテストデバイスを用いた以外は、実験例１と同様の操作で、インフルエンザウイルスの測定を行った。判定の結果を表４に示す。 Experimental Examples 13-18 Measurement of Influenza Virus Influenza virus was measured in the same manner as in Experimental Example 1, except that the test devices of Comparative Examples 7-12 were used. Table 4 shows the result of the determination.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　実験例５～１８の結果から明らかなように、膜可溶化剤のなかでも、特定の膜可溶化剤のみが、非特異反応を抑制できることがわかった。また、非特異反応の抑制剤として一般的に用いられているタンパク質やアミノ酸を使用しても、ブランク測定において「陽性」反応が発現してしまい、特異的かつ確度の高い測定ができないことがわかった。すなわち、特定の膜可溶化剤を、抗体等の特異的結合物質と混合させ、その混合液を非吸収性担体に塗布することによってのみ、非特異反応を抑制できることがわかった。 As is clear from the results of Experimental Examples 5 to 18, it was found that only specific membrane solubilizers can suppress non-specific reactions among membrane solubilizers. In addition, even when using proteins and amino acids commonly used as inhibitors of non-specific reactions, it was found that a “positive” reaction was expressed in the blank measurement, and specific and highly accurate measurement could not be performed. It was. That is, it was found that a non-specific reaction can be suppressed only by mixing a specific membrane solubilizer with a specific binding substance such as an antibody and applying the mixture to a non-absorbable carrier.
　以上の結果から明らかなように、本発明の固定化方法によって得られる非吸収性担体を用い、例えばインフルエンザウイルス等を測定すれば、非特異反応を抑制することができ、精度よく測定対象物質を検出できることがわかった。 As is clear from the above results, if 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.
　本発明の特異的結合物質の固定化方法を用いて得られた非吸収性担体を、例えばインフルエンザウイルス等の測定に用いることにより、非特異反応を抑制することができ、精度よく「陽性」または「陰性」を判定することが可能となる。 By using 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”.
１：平底マイクロプレート
２：ウェルの平底面
３，６，８，８'，１１，１１'，１５：特異的結合物質で被覆されている部分
４：粒子
５：直方体型透明容器
７，９：長方形の板状体
１０：流路（溝）
１２：カバー
１３：ポリスチレン板状体（テストデバイス）
１４，１４'：ポリスチレン板
１６：ポリスチレン基板 1: Flat bottom microplate 2: Flat bottom surface of well 3, 6, 8, 8 ', 11, 11', 15: Part coated with specific binding substance 4: Particle 5: Cuboid transparent container 7, 9: Rectangular plate 10: channel (groove)
12: Cover 13: Polystyrene plate (test device)
14, 14 ': Polystyrene plate 16: Polystyrene substrate

Claims (16)

  1. 測定対象物質に特異的に結合する物質（特異的結合物質）と、ｎ-オクチル-β-Ｄ-グルコシド、ｎ-オクチル-β-Ｄ-チオグルコシド、ｎ-ドデシル-β-Ｄ-マルトシド、ｎ-ノニル-β-Ｄ-チオマルトシドおよびｎ-オクタノイル-Ｎ-メチル-Ｄ-グルカミンからなる群から選ばれる膜可溶化剤とをあらかじめ混合し、該特異的結合物質と該膜可溶化剤とを含む混合液を非吸収性担体の表面に塗布して、該表面に特異的結合物質を固定化することを特徴とする、特異的結合物質の固定化方法。 A substance that specifically binds to the substance to be measured (specific binding substance), n-octyl-β-D-glucoside, n-octyl-β-D-thioglucoside, n-dodecyl-β-D-maltoside, n A membrane solubilizing agent selected from the group consisting of -nonyl-β-D-thiomaltoside and n-octanoyl-N-methyl-D-glucamine is preliminarily mixed, and the specific binding substance and the membrane solubilizing agent are included. A method for immobilizing a specific binding substance, which comprises applying a mixed solution to a surface of a non-absorbable carrier and immobilizing a specific binding substance on the surface.
  2. 前記混合液を前記非吸収性担体の表面に塗布した後に、さらにブロッキング処理を行う、請求項１に記載の固定化方法。 The immobilization method according to claim 1, wherein a blocking treatment is further performed after the mixed solution is applied to a surface of the non-absorbable carrier.
  3. 前記非吸収性担体が、プラスチック担体である、請求項１に記載の固定化方法。 The immobilization method according to claim 1, wherein the non-absorbable carrier is a plastic carrier.
  4. 前記プラスチック担体が、ポリエチレン製、ポリプロピレン製またはポリスチレン製のものである、請求項３に記載の固定化方法。 The immobilization method according to claim 3, wherein the plastic carrier is made of polyethylene, polypropylene, or polystyrene.
  5. 前記膜可溶化剤が、ｎ-オクチル-β-Ｄ-グルコシド、ｎ-オクチル-β-Ｄ-チオグルコシドおよびｎ-オクタノイル-Ｎ-メチル-Ｄ-グルカミンからなる群から選ばれるものである、請求項１に記載の固定化方法。 The membrane solubilizer is selected from the group consisting of n-octyl-β-D-glucoside, n-octyl-β-D-thioglucoside and n-octanoyl-N-methyl-D-glucamine. Item 2. The immobilization method according to Item 1.
  6. 前記混合液中の前記膜可溶化剤の濃度が、０．００１～０．１重量％である、請求項１に記載の固定化方法。 The immobilization method according to claim 1, wherein the concentration of the membrane solubilizer in the mixed solution is 0.001 to 0.1 wt%.
  7. 前記特異的結合物質が、抗原または抗体である、請求項１に記載の固定化方法。 The immobilization method according to claim 1, wherein the specific binding substance is an antigen or an antibody.
  8. 前記固定化が、圧電素子を備えるヘッドを用いて前記混合液を前記非吸収性担体の表面に塗布することにより行われる、請求項１に記載の固定化方法。 The immobilization method according to claim 1, wherein the immobilization is performed by applying the mixed liquid onto a surface of the non-absorbable carrier using a head including a piezoelectric element.
  9. 測定対象物質に特異的に結合する物質（特異的結合物質）と、ｎ-オクチル-β-Ｄ-グルコシド、ｎ-オクチル-β-Ｄ-チオグルコシド、ｎ-ドデシル-β-Ｄ-マルトシド、ｎ-ノニル-β-Ｄ-チオマルトシドおよびｎ-オクタノイル-Ｎ-メチル-Ｄ-グルカミンからなる群から選ばれる膜可溶化剤とをあらかじめ混合し、該特異的結合物質と該膜可溶化剤とを含む混合液を非吸収性担体の表面に塗布することによって得られる、該表面に特異的結合物質が固定化された非吸収性担体。 A substance that specifically binds to the substance to be measured (specific binding substance), n-octyl-β-D-glucoside, n-octyl-β-D-thioglucoside, n-dodecyl-β-D-maltoside, n A membrane solubilizing agent selected from the group consisting of -nonyl-β-D-thiomaltoside and n-octanoyl-N-methyl-D-glucamine is preliminarily mixed, and the specific binding substance and the membrane solubilizing agent are included. A non-absorbable carrier obtained by applying a mixed solution to the surface of a non-absorbable carrier, wherein a specific binding substance is immobilized on the surface.
  10. 前記混合液を前記非吸収性担体の表面に塗布した後に、さらにブロッキング処理を行うことによって得られるものである、請求項９に記載の担体。 The carrier according to claim 9, which is obtained by further performing a blocking treatment after applying the mixed solution to the surface of the non-absorbable carrier.
  11. 前記非吸収性担体の表面が、特異的結合物質により部分的に被覆されている、請求項９に記載の担体。 The carrier according to claim 9, wherein the surface of the non-absorbable carrier is partially coated with a specific binding substance.
  12. 前記非吸収性担体が、板状体である、請求項９に記載の担体。 The carrier according to claim 9, wherein the non-absorbable carrier is a plate-like body.
  13. 前記非吸収性担体が、平底面を有する凹部を少なくとも１つ備えた容器であって、該平底面が特異的結合物質により部分的に被覆されている、請求項９に記載の担体。 The carrier according to claim 9, wherein the non-absorbable carrier is a container having at least one recess having a flat bottom surface, and the flat bottom surface is partially covered with a specific binding substance.
  14. 前記非吸収性担体が、プラスチック担体である、請求項９に記載の担体。 The carrier according to claim 9, wherein the non-absorbable carrier is a plastic carrier.
  15. 前記プラスチック担体が、ポリエチレン製、ポリプロピレン製またはポリスチレン製のものである、請求項１４に記載の担体。 The carrier according to claim 14, wherein the plastic carrier is made of polyethylene, polypropylene or polystyrene.
  16. 請求項９乃至請求項１５の非吸収性担体を用いることを特徴とする、試料中の測定対象物質の測定方法。 A method for measuring a substance to be measured in a sample, wherein the nonabsorbable carrier according to claim 9 is used.
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