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WO2006088192A1 - Support destine a l'utilisation analytique et procede de determination utilisant ce support - Google Patents

Support destine a l'utilisation analytique et procede de determination utilisant ce support Download PDF

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Publication number
WO2006088192A1
WO2006088192A1 PCT/JP2006/302993 JP2006302993W WO2006088192A1 WO 2006088192 A1 WO2006088192 A1 WO 2006088192A1 JP 2006302993 W JP2006302993 W JP 2006302993W WO 2006088192 A1 WO2006088192 A1 WO 2006088192A1
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WO
WIPO (PCT)
Prior art keywords
carrier
binding
measurement
capture molecule
measurement object
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2006/302993
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English (en)
Japanese (ja)
Inventor
Takeshi Serizawa
Hisao Matsuno
Tatsuya Shinoda
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Tokyo NUC
Minaris Medical Co Ltd
Original Assignee
Kyowa Medex Co Ltd
University of Tokyo NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyowa Medex Co Ltd, University of Tokyo NUC filed Critical Kyowa Medex Co Ltd
Priority to JP2007503775A priority Critical patent/JPWO2006088192A1/ja
Publication of WO2006088192A1 publication Critical patent/WO2006088192A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin

Definitions

  • the present invention relates to a carrier for measuring a measurement object and a capture molecule-bound carrier, a measurement reagent containing the carrier or the capture molecule-bound carrier, a method for producing the carrier and the capture molecule-bound carrier, the carrier and
  • the present invention relates to a method for measuring an object to be measured using a capture molecule binding carrier.
  • a measurement method using a specific reaction such as an antigen-antibody reaction and a measurement method using an enzyme reaction are known.
  • a substance such as an antibody that specifically binds to a measurement object such as an antigen is immobilized on a support, and the measurement object and a substance that specifically binds to the measurement object are bound and bound.
  • Measurement object can be measured by various methods, for example, the method of measuring the amount of bound enzyme by binding antibody-enzyme complex (ELISA method), the method of measuring the change in dielectric constant (surface plasmon resonance method), the change of weight It is measured by measuring the amount of heat (quartz crystal method) and measuring the amount of generated heat (trace differential calorimetry) (see, for example, Patent Documents 1 and 2).
  • ELISA method the method of measuring the amount of bound enzyme by binding antibody-enzyme complex
  • surface plasmon resonance method surface plasmon resonance method
  • the change of weight It is measured by measuring the amount of heat (quartz crystal method) and measuring the amount of generated heat (trace differential calorimetry)
  • a substance such as an enzyme that specifically binds the measurement target is fixed to the support, the measurement target is bound, and a reaction product generated by the reaction between the measurement target and the enzyme or This can be done by detecting changes in the amount of material caused by the conversion, for example optical changes.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2004-37209
  • Patent Document 2 Japanese Patent Application Laid-Open No. 2004-117026
  • Patent Document 3 Japanese Patent Application Laid-Open No. 2004-340766.
  • the present invention has been made in view of the above circumstances, and a carrier for measuring a measurement object in a sample and a capture molecule-bound carrier, a measurement method using the carrier and the capture molecule-bound carrier, the carrier or
  • An object of the present invention is to provide a reagent for measuring a measurement object containing the capture molecule-binding carrier, or a method for producing the carrier or the capture molecule-binding carrier.
  • the present inventors formed an aggregate of two or more polymers having different stereoregularities on the surface of the support, and distributed on the surface of the aggregate.
  • the inventors have found that the above problems can be solved by immobilizing a substance for binding a child and, if necessary, a capturing molecule, and have completed the present invention.
  • the substance for molecular binding can be easily fixed at a high density and without a crosslinking agent, and denaturation of the substance for molecular binding is suppressed even after long-term storage. Can be suppressed, and accurate and reproducible measurement can be performed.
  • the present invention relates to the following:!
  • An object to be measured in a sample comprising a support having an aggregate region of two or more polymers having different stereoregularities on the surface, and a molecular binding substance fixed to the aggregate region of the support.
  • Carrier for measuring objects comprising a support having an aggregate region of two or more polymers having different stereoregularities on the surface, and a molecular binding substance fixed to the aggregate region of the support.
  • the carrier according to 1 above which comprises an aggregate in which the aggregate forms a sterically complementary association.
  • the carrier according to 1 or 2 above comprising two or more polymer forces having stereoregularity S, a syndiotactic polymer and a isotactic polymer.
  • polymetatalate is polymethyl metatalylate, polyethyl metatalylate, polyisopropyl metatalylate or poly n-propyl metatalylate.
  • a capture molecule-bound carrier for measuring a measurement object in a sample wherein a capture molecule is bound to the carrier according to 10 or 11 via the capture molecule-binding protein.
  • a reagent for measuring a measurement object comprising the carrier according to any one of the above:! To 12.
  • a measurement object measuring kit comprising the carrier according to any one of the above:! To 12.
  • a kit for measuring an object to be measured in a sample comprising a reagent including a support having an aggregate region of two or more polymers having different stereoregularities on the surface, and a reagent including a molecule binding substance.
  • a step of bringing the carrier described in 10 or 11 above into contact with a capture molecule to form a capture molecule-bound carrier, contacting the capture molecule-bound carrier with a measurement object and passing the capture molecule through the capture molecule The step of binding the carrier to the carrier, and measuring the measurement object bound to the carrier
  • the measuring method of the measuring object in a sample including the process to perform.
  • a step of bringing a capture molecule into contact with a measurement object to form a measurement object-bound capture molecule; contacting the support according to 10 or 11 above with a measurement object-bound capture molecule to bring the measurement object into a carrier A method for measuring a measurement object in a sample, comprising: a step of binding to a carrier; and a step of measuring a measurement object bound to the carrier.
  • a step of immobilizing a molecular binding substance on an aggregate region of a support having two or more polymer aggregate regions having different stereoregularities on the surface and a step of binding a capture molecule to the molecular binding material
  • a method for producing a capture molecule-binding carrier for measuring a measurement object in a sample A method for producing a capture molecule-binding carrier for measuring a measurement object in a sample.
  • Different materials used for a carrier for measuring a measurement object in a sample by immobilizing a molecular binding substance in an aggregate region of two or more polymers having different stereoregularities on a support surface A polymer adhering support having a surface of an aggregate region of two or more polymers having stereoregularity.
  • a carrier having improved storage stability, a capture molecule-bound carrier and a measurement reagent, a method for measuring a measurement object in a measurement sample with improved measurement accuracy or sensitivity, and a simple carrier and capture molecule A method of manufacturing a binding carrier is provided.
  • FIG. 1 shows a carrier when a capture molecule-binding protein is used as a molecule-binding substance.
  • FIG. 3 is a schematic view showing one embodiment of a body formation reaction, a capture molecule-bound carrier formation reaction, and a measurement object binding reaction.
  • FIG. 2 is a conceptual diagram showing one embodiment of a carrier formation reaction when an enzyme is used as a molecule binding substance and a reaction between the enzyme on the carrier and a measurement object (substrate). .
  • FIG. 3 shows a calibration curve showing the relationship between the amount of HSA antigen binding and the concentration of HSA antigen.
  • ⁇ represents the amount of HSA antigen binding in Example 1
  • the mouth represents Comparative Example 6
  • ⁇ represents Comparative Example 9.
  • FIG. 4 is a graph showing the measurement results of the fixed amount of ⁇ -galatatosidase using a QCM chip.
  • ⁇ represents the amount of binding of / 3_galactosidase of Example 4 and ⁇ of Comparative Example 12, respectively.
  • FIG. 5 is a graph showing an evaluation of the activity of ⁇ -galatatosidase immobilized on a maxisorp plate.
  • the bold line O represents the results of Example 5
  • the thin line port represents Comparative Example 13
  • the broken line represents the results of Comparative Example 14.
  • Fig. 6 is a graph showing the activity evaluation of ⁇ -galatatosidase immobilized on a maxi-soap plate for 7 days.
  • the bold line O represents the results for Example 5
  • the thin line port represents Comparative Example 13
  • the wavy line ⁇ represents the results for Comparative Example 14.
  • the support used in the present invention is not particularly limited as long as it can be used as a solid phase, and is used for measurement, diagnosis, examination, etc. by a method using specific binding such as antigen-antibody reaction, enzyme reaction, etc.
  • a support made of a material to be used can be used, and is appropriately selected according to the measurement method of the solid-phased measurement object. For example, when measurement is performed by a spectroscopic technique, a support material is selected according to the light transmittance used.
  • the support material includes, but is not limited to, quartz, polymer, glass, ceramics, and the like.
  • As the crystal various crystal forms that can be used as a crystal resonator can be mentioned. Depending on the application, various known cuts may be applied.
  • Polymers for the support material include, for example, polymethyl methacrylate, polyethyl methacrylate, poly ⁇ -propyl methacrylate, polyisopropyl methacrylate, poly ⁇ -butyl methacrylate, polyisobutyl methacrylate.
  • Polymethacrylates such as polymethacrylic acid, for example polymethyl acrylate
  • Polyacrylates such as acrylate, polyethyl acrylate, poly n-butyl acrylate, polyisobutyl acrylate, polyacrylic acid, polycarbonate such as bisphenol A polycarbonate, polyacrylonitrile, polystyrene, polyethylene, polypropylene, ABS, etc.
  • Examples of the glass include soda lime glass, cowing glass, and quartz glass.
  • the support may be appropriately provided with a metal thin film (for example, a gold thin film), an electrode, a pre-coating, or the like depending on the purpose of measurement of the measurement object.
  • a metal thin film for example, a gold thin film
  • an electrode for example, a gold thin film
  • a pre-coating for example, a pre-coating
  • the support is, for example, a measurement plate used for immunoassay such as 96 or 384 well plate, beads such as Luminex beads, for example, fine particles, magnetic particles, gold used in immunoturbidimetric methods, etc.
  • Fine particles such as colloids, for example, membranes used for immunochromatography and Western blotting, for example, quartz crystal chips, chips used for surface plasmon resonance, chips having a plurality of antibody binding regions on one flat plate
  • a chip such as a biochip constructed by integrating a flow path, a reaction field and a detection part on a support by microfabrication technology, such as a protein system.
  • Array Japanese Patent Laid-Open No.
  • the aggregate of two or more polymers having different stereoregularity is an aggregate of two or more polymers, and all or a part of the two or more polymers is each An aggregate having different stereoregularity.
  • the polymer may be a block in a block copolymer.
  • the aggregate of two or more polymers having different stereoregularity may be formed by associating two or more blocks having different stereoregularity in one molecule. That is, in the aggregate, in a polymer having two or more blocks having stereoregularity and other blocks having different stereoregularity, a molecule formed by association of these two or more blocks. Internal associations are also included.
  • the monomer that is a raw material of the polymer used in the polymer aggregate is a known one. Can be selected.
  • the monomers are propylene, 1-butene, 1-pentene, 3-methyl-1-butene, 4-methyl-1 pentene, 4,4 dimethyl-1 pentene, and other alkenes, styrene, 2-methylstyrene, 3-methylstyrene, 2 , 4 Dimethylstyrene, 2,5_dimethylstyrene, 3,4_dimethylstyrene, 3,5_dimethylstyrene, butadienes such as butadiene and isoprene, methacrylic acid, methyl methacrylate, butyl acrylate, N-isopropyl acrylamide, N, N-diisopropyl acrylamide, acrylic acid derivatives such as acrylonitrile or methacrylic acid derivatives, butyl ethers such as butyl butyl ether,
  • the stereoregularity of the polymer can be controlled by appropriately selecting the experimental conditions such as catalyst, temperature, solvent, etc., using the above monomers as raw materials. Any known method can be used as the polymerization method. Examples include anion polymerization, coordination anion polymerization, and radical polymerization. It is also possible to separate a polymer having stereoregularity by separating a polymer obtained by polymerizing the above monomer as a raw material by an appropriate known means.
  • the stereoregularity includes the regularity regarding the arrangement of substituents with respect to the polymer main chain and the regularity obtained by using only one of the enantiomers as a monomer.
  • a polymer having a substituent on either side of the polymer main chain is referred to as a isotactic polymer (hereinafter abbreviated as it-polymer), and the substituent is separated from the polymer main chain.
  • the polymer that is regularly replaced by this is called syndiotactic polymer (hereinafter abbreviated as st-polymer).
  • st-polymer syndiotactic polymer
  • a polymer in which both properties are irregularly mixed is called atactic polymer.
  • Examples of monomers that give it-polymers include, for example, propylene, 1-butene, 1_pentene, 3_methyl_1-butene, 4_methyl_1_pentene, 4,4-dimethyl mono 1_ Pentene, styrene, 2-methylstyrene, 3-methylstyrene, 2,4_dimethylstyrene, 2,5_dimethylstyrene, 3,4_dimethylstyrene, 3,5_dimethylstyrene, butadiene Strength including, but not limited to, methacrylic acid derivatives such as styrene, methacrylic acid, and methyl methacrylate, butyl acrylate, butyl vinyl ether, N, N diisopropylacrylamide, propylene oxide, and cetaldehyde.
  • methacrylic acid derivatives such as styrene, methacrylic acid, and methyl methacrylate, butyl acrylate,
  • Examples of the monomer that provides the st-polymer include, but are not limited to, force S, such as methacrylic acid derivatives such as butadiene and methylol methacrylate, N_isopropylacrylamide, and the like.
  • the polymer having stereoregularity in the present invention is not limited to the one having ideal stereoregularity, and a certain regularity that may be a somewhat disordered structure in the molecule continues to some extent. It can be a polymer like a candy.
  • the it-polymer isotacticity (% mm; triplet display) is generally 70 or more, preferably 80 or more, more preferably 90 or more.
  • the syndiotacticity (% rr; triplet display) is generally 70 or more, preferably 80 or more, more preferably 90 or more.
  • the number average molecular weight (Mn) of the polymer is generally 1,000 or more, preferably 5,000 or more, and more preferably 10,000 or more.
  • the weight average molecular weight (Mw) of the polymer is generally 1,000 or more, preferably 5,000 or more, and more preferably 10,000 or more.
  • the ratio of the weight average molecular weight to the number average molecular weight (Mw / Mn) is 1 or more, generally 5 or less, preferably 2 or less, more preferably 1.5 or less.
  • the polymer having stereoregularity in the present invention includes a polymer obtained by polymerizing one kind of enantiomer as a polymerization unit.
  • Examples of the combination of the polymer and the polymer having a different stereoregularity from the polymer include a combination of poly D lactic acid and poly L lactic acid.
  • an aggregate in which the polymers are associated in a sterically complementary manner is preferable.
  • an aggregate in which polymers are sterically complementary is called a stereo complex.
  • Stereocomplementary association means that the free energy of the polymer forming the stereocomplex associates so as to stabilize at a minimum or local minimum, and that the polymer is associated in a manner that the polymer is closely packed. including.
  • Examples of the polymer forming the stereo complex include polymetatalylate and polylactic acid.
  • the polymetatalylate used in the present invention is not limited as long as it is a polymer obtained by polymerizing a monomer selected from the group consisting of methacrylic acid and polymethacrylic acid ester, and homopolymer having one type of polymerization unit. Even if it is a polymer, the polymerization unit may be a plurality of types of copolymers.
  • Specific examples of the polymetatalate of the present invention include polymethyl metatalate (hereinafter abbreviated as PMMA), polyethyl methacrylate (hereinafter abbreviated as PEMA), poly-n-propyl metatalylate, Forces including, but not limited to, polyisopropyl methacrylate.
  • PMMA polymethyl metatalate
  • PEMA polyethyl methacrylate
  • Poly-n-propyl metatalylate Forces including, but not limited to, polyisopropyl methacrylate.
  • Polymetatalylate is available from Polymer Source, Inc.,
  • stereocomplexes include a structure formed between a polymer having stereoregularity and a polymer having a different stereoregularity from the polymer and having various length ratios with respect to the length of the polymer.
  • the polymer is a structure other than the structure in which a helix is formed by van der Waals (hereinafter referred to as a helix structure) and the structure in which a helix is formed on the basis of van der Waals aggregation. Structure (hereinafter referred to as van der Waals structure).
  • a structure that forms a helix specifically, a structure that forms a double helix (hereinafter referred to as a double helix structure: Serizawa et al., J. Am. Chem. Soc. 2000, Vol. 122, ⁇ ⁇ 1891 But are not limited to these.
  • the double helix structure is a structure in which an it-polymer is surrounded by a st-polymer that is twice as long, and Van der Waals forces act between them.
  • the it-polymer and st-polymer in the double helix structure may be the same or different.
  • PMMA can be used for both it-polymer and st-polymer
  • PMMA can be used for one of it-polymer and st-polymer
  • PEMA for the other.
  • Van der Waals structure a structure in which it-polymer and st-polymer of the same length are associated is known.
  • the method of forming the stereo complex can be made by dissolving and mixing the polymer to be associated with a suitable solvent, and if necessary, it can be made by separating the desired stereocomplex.
  • the alternating layer (layer-by-layer) method (hereinafter abbreviated as LbL method) described in J. Am. Chem. Soc, 2000 Vol. 122, p.1891 Is used. This method uses a syndiotactic support.
  • At least one stereocomplex layer formation cycle including the step of immersing in the tick polymetatalylate solution and the step of immersing the support in the isotactic polymetatalylate solution to form a polymer film containing the stereocomplex.
  • it-polymer and st-polymer were separately dissolved in an appropriate solvent (hereinafter referred to as a film-forming solvent), and the support was immersed in the it-polymer solution to adsorb the it-polymer.
  • the number of cycles there is no limit to the number of cycles, that is, the number of stereocomplex layers.
  • the number of cycles is 2 times or more, preferably 5 times or more.
  • the upper limit of the number of cycles is not particularly limited, but it is 50 times or less, preferably 30 times or less from an economic viewpoint.
  • the film-forming solvent can be selected from water, a polar organic solvent, or a mixture thereof.
  • the polar organic solvent include acetonitrile, dimethylformamide, acetone, alcohols, dimethyl sulfoxide, dioxane and the like.
  • the water may contain a buffer.
  • the polymer concentration is not limited as long as it is suitable for forming a film, but is generally 0.1 mg / mL or more, preferably 0.5 mg / mL or more, more preferably 1 mg / mL or more, and generally 20 mg. / mL or less, preferably 10 mg / mL or less, more preferably:! to 5 mg / mL or less.
  • the film formation temperature is generally 4 ° C or higher, preferably 10 ° C or higher, more preferably 20 ° C or higher, and generally 50 ° C or lower, preferably 40 ° C or lower, more preferably 30 ° C. It is below ° C.
  • the immersion time is generally 1 minute or longer, preferably 3 minutes or longer, more preferably 5 minutes or longer. There is no particular upper limit, but it is preferably 60 minutes or less, more preferably 30 minutes or less.
  • an intramolecular aggregate since it can be easily formed into a form other than film formation, it is also suitable for use with beads or the like.
  • One embodiment of the polymer that forms intramolecular aggregates is a block copolymer having a isotactic block and a syndiotactic block, a block obtained by polymerizing one type of enantiomer as a polymerization unit, and another type of mirror image And a block copolymer having a polymerized block having an isomer as a polymerized unit.
  • the block copolymer may be produced by linking a isotactic block and a syndiotactic block directly or via a linker, or by block polymerization. Specific examples include the following methods. When synthesizing a stereoregular polymetatalylate by low-temperature living anion polymerization, a polymer having a hydroxyl group, a carboxynole group, an amino group, etc. at one end can be synthesized by appropriately selecting a reaction terminator. A block copolymer in which an isotactic block and a syndiotactic block coexist in one molecule can be synthesized by condensation by ester or amide formation between these end groups or bonding to an appropriate linker. These form hairpins by interaction and form stable intramolecular stereocomplexes.
  • the block copolymer may be produced by polymerizing another type of enantiomer following the block polymerized using one type of enantiomer as a polymerization unit.
  • a block obtained by polymerizing one kind of isomer as a polymerized unit and a block polymerized by polymerizing another kind of enantiomer as a polymerized unit may be produced directly or through a linker.
  • a block copolymer containing a lactic acid block and a poly L-lactic acid block can be synthesized.
  • synthesizing a poly D-lactic acid block and a poly L-lactic acid block by selecting an appropriate reaction terminator as described above, As a result of the condensation reaction or the coupling reaction similar to the above, a block copolymer can be obtained.
  • the block copolymer forms a stable intramolecular stereocomplex by intramolecular interaction.
  • the molecular binding substance in the present invention means a substance that can be fixed to an aggregate region of a support having on its surface an aggregate region of two or more polymers having different stereoregularities. To do.
  • the substance for molecular binding immobilized on the aggregate region of the support has affinity for the substance to be measured and can be directly bound to the object to be measured, or in some cases, the capture molecule described below. Can be combined with. In such a case, the measurement object can be bound to the molecule binding substance via the capture molecule.
  • Examples of such molecular binding substances include proteins for binding capture molecules such as protein A, protein G or protein L, Fc receptor, or a combination thereof, and molecular binding substances that can directly bind to the analyte. Examples include enzymes.
  • an enzyme for example, an oxidoreductase, a transferase, a hydrolase, a dehydrogenase, an isomerase, a synthase and the like can be preferably used.
  • oxidoreductase those using CH-OH as a donor, those using an aldehyde or oxo group as a donor, those using CH-CH as a donor, those using CH-NH as a donor
  • NADH reduced nicotinamide adenine dinucleotide
  • NADPH reduced nicotinamide adenine dinucleotide phosphate
  • Examples of those using CH-OH as a donor include alcohol dehydrogenase, dalcose-6_phosphate dehydrogenase, glycerol dehydrogenase, and glycerol-3. Phosphate dehydrogenase, 3 ⁇ -hydroxysteroid dehydrogenase, 7 ⁇ -hydrogenate dehydrogenase, L-lactate dehydrogenase, 3-hydroxybutyrate dehydrogenase, pyranose oxidase, glycerol oxidase, alcohol oxidase, choline oxidase, galactose Xidase, Gnorecose Oxidase, Cholesterol Oxidase, L- ⁇ -Glycetophosphate Oxidase, Lactate Oxidase, D-Lactate Dehydrogenase, Maleate Dehydrogenase, Isopropyl Maleate Dehydrogenase, Phosphodarconate
  • Examples of the donor having an aldehyde or oxo group include pyruvate oxidase, glycealdehyde _3_phosphate dehydrogenase, xanthine oxidase, pyruvate dehydrogenase, formaldehyde dehydrogenase and the like.
  • CH-CH as a donor include acyl-CoA dehydrogenase, bilirubinoxidase and the like.
  • Examples of those having CH-NH as a donor include glutamate synthetase, alanine dehydrogenase, tyramine oxidase, aminoxidase, putrescine oxidase, glutamate dehydrogenase, leucine dehydrogenase, L amino acid oxidase, cytochrome P450 Etc.
  • Examples of those using CH-NH as a donor include sarcosine oxidase, sarcosine dehydrogenase, ⁇ 1-piperidin 2-power rubonic acid reductase, and the like.
  • NADH or NADPH examples include glutathione reductase, diaphorase, NADH-flavin mononucleotide (hereinafter abbreviated as FMN) oxide reductase, NADPH-FMN oxidoreductase, dihydrolipoamide reductase, and the like.
  • FMN NADH-flavin mononucleotide
  • NADPH-FMN oxidoreductase dihydrolipoamide reductase, and the like.
  • Examples of those using a nitrogen compound as a donor include uricase.
  • Examples of those using diphenol or related compounds as a donor include ascorbic acid. And oxidase.
  • Examples of those having hydrogen peroxide as an acceptor include peroxidase, gnorethion peroxidase, and the like.
  • Examples of compounds that take up molecular oxygen and act on a pair of donors include p-hydroxybenzoate 4_monooxygenase, salicylic acid 1_monooxygenase, and steroid 11 ⁇ -monooxy. Examples include shigenase, phenylalanin 4_monoxygenase, dopamine j3 -monooxygenase.
  • Examples of the substance that acts as a receptor for superoxide include superoxide dismutase and the like.
  • Examples of donors that contain sulfur-containing groups include sulfite reductase, hypotaurine dehydrogenase, sulfite oxidase, thioloxidase, glutathione-homocystine transhydrogenase, protein monodisulfide reductase, glutathione cysteine transhydrogenase. And dartathione dehydrogenase.
  • heme as a donor examples include cytochrome coxidase, nitrate reductase and the like.
  • Examples of hydrogen molecule donors include hydrogen dehydrogenase and cytochrome c3 hydrogenase.
  • Examples of those that take up molecular oxygen and act on a single donor include, for example, catechol 1,2-dioxygenase, catechol 2,3-dioxygenase, tryptophan 2,3 dioxygenase, lipoxygenase, aspirolbe 2,3 Dioxygenase, Arginine 2-Monoxygenase, Lysine 2-Monoxygenase, Tryptophan 2_Monoxygenase, Lactic acid 2_Monoxygenase, Renilla Cipherin 2_Mono Oxygenase, cigarette luciferin 2_monoxygenase, firefly luciferin 2-monooxygenase, and the like.
  • Examples of those acting on CH group or CH group include xanthine dehydrogenase, nicotinate dehydrogenase and the like.
  • ferrodoxine monoxide type NAD examples include reductase and ferrodoxine monoxide NADP reductase.
  • Examples of those using reduced flavodoxin as a donor include nitrogenase and the like.
  • Examples of the transferase include those that transfer one carbon group, those that transfer an aldehyde or ketone, those that transfer an alkyl group or aryl group other than a methyl transferase, an acyl transferase, a glycosyl transferase, or a methyl group. , Those that transfer groups containing nitrogen, those that transfer groups containing phosphorus, and those that transfer groups containing sulfur.
  • Examples of those that transfer one carbon group include nicotinamide methyltransferase, histamine methyltransferase, homocystine methyltransferase, and the like.
  • Examples of those that transfer aldehyde or ketone include transketolase and transaldolase.
  • acyl transferase examples include amino acid acetyl transferase, darcosamine acetyl transferase, choline acetyl transferase, acetyl acetyl transferase phosphatase, acetyl acetyl CoA transferase, galactoside acetyl transferase, diacyl glycerol acyl transferase, lysolecithin
  • Examples include acyl transferase, glutamate acetyl transferase, histone acetyl transferase, peptidyl transferase, citrate synthase, ⁇ -glutamino retransferase, phosphotransacetylase, transdaltaminase and the like.
  • glycosyltransferases include phosphorylase, purine nucleotide phosphorylase, sucrose phosphorylase, manoletos phosphorylase, glycogen synthase, cenorelose synthase, chitin synthase, UDP gnolecronosinotransferase, U DP Examples include galactose monocollagen galactosyltransferase and GDP fucose monosaccharide protein fucosyltransferase.
  • Examples of those that transfer an alkyl group or an aryl group other than a methyl group include, for example, dimethylaryltransferase, terpenoid monotransferase, aminopropyltransferase, farnesyltransferase, phosphoserine sulfide. Lilase etc. S can give.
  • Examples of those capable of transferring a nitrogen-containing group include aspartate aminotransferase, alanine aminotransferase, diamino acid aminotransferase, and acetylnitronitine aminotransferase.
  • Examples of those that transfer phosphorus-containing groups include creatine kinase, myokinase, hexokinase, phosphodalcomtase, phosphoglyceromutase, pyruvate kinase, glyceronorekinase, acetate kinase, phosphofunolectokinase, phosphoglycerase Tokinase, Polynucleotide phosphorylase, Phospholipase A2, Adenosine kinase, Thymidine kinase, Protein kinase, Adenylate kinase, Nucleoside phosphate kinase, Nucleoside diphosphate kinase, Nicotinamide mononucleotide adenylyltransferase, FMN adenylyltransferase, DNA Nucleotidyl transferase, RNA
  • Examples of those capable of transferring a group containing sulfur include allyl sulfotransferase, estrone sulfotransferase, chondroitin sulfotransferase, and acetyl Co A transferase.
  • Hydrolytic enzymes include those acting on ester bonds, those acting on glycosyl compounds, those acting on ether bonds, those acting on peptide bonds, those acting on CN bonds other than peptides, acid anhydrides Those that act on compounds, those that act on CC bonds, those that act on halides, those that act on P—N bonds, and those that act on S—N bonds.
  • Examples of substances that act on ester bonds include carboxyesterase, aryl esterase, lysophospholinocase, cholesterol monoreesterase, phosphorinocase A2, phospholipase c , phospholipase 0, sphingomyelinase, alkaline phosphatase Acid phosphatase, lipoprotein lipase, 5 'nucleotidase, 3, nucleotidase, gnolecose 6-phosphate phosphatase, funolectose bisphosphatase, phosphodiesterase, allyl sulfatase, sterol sulfatase, exodeoxy Ribonuclease, exoribonuclease, endo-deoxyribonuclease, endoribonuclease There is a case.
  • glycosyl compounds examples include ⁇ -amylase, amylase, cellulase, dextranase, chitinase, chitosanase, lysozyme, O dalcosidase, / 3- dalcosidase, / 3_galactosidase, j3-darc mutidase , ⁇ -fucosidase, j3— ⁇ -acetylyldarcosaminidase, endo 1,3_ / 3-glucanase, endo 1,3- ⁇ -galactanase, endo-1,4- ⁇ -galactanase, gnolecan 1, 6 -a-Dalcosidase, neuraminidase, invertase, nucleosidase, isopranulase and the like.
  • Examples of those that act on the ether bond include adenosylmethionine hydrolase.
  • agents that act on peptide bonds include aminopeptidases, carboxypeptidases, prolylaminopeptidases, purine iminopeptidases, pyroglutamyl peptidases, dipeptidinorepeptidases, dipeptidylgrecanoloxypeptides.
  • agents that act on peptide bonds include peptidase, trypsin, chymotrypsin, thrombin, plasmin, blood coagulation factor, kallikrein, elastase, cathebucin, papain, phytin, bromelain, chymopapain, cysteine proteinase, pepsin, chymosin, aspartic proteinase, collagenase and the like.
  • Examples of those that act on C—N bonds other than peptides include urease, creatinine amide hydrolase, creatinine deaminase, creatinase, creatininase, asparaginase, glutaminase, aminoacylase, N acetylylmuramoylala Ninamidase and the like.
  • Examples of substances that act on acid anhydrides include avilase and ATP pyrophosphatase.
  • Examples of the substance that acts on the C—C bond include oxaloacetase and the like.
  • Examples of substances that act on halides include alkylhalidase, haloacetate dehalogenase, and fluoroacetate dehalogenase.
  • Examples of the substance acting on the PN bond include phosphoamidase.
  • Examples of the substance that acts on the S—N bond include sulfodarcosamine sulfohydrolase, etc. Can be given.
  • desorption enzymes include those that act on carbon-carbon bonds, those that act on carbon-oxygen bonds, those that act on carbon-nitrogen bonds, those that act on carbon-sulfur bonds, and those that act on phosphate bonds. Is given.
  • Examples of substances that act on carbon-carbon bonds include N-acetylneuraminate aldolase, oxalate acetic acid decarboxylase, pyruvate decarboxylase, gnoletamic acid decarboxylase, ornithine decarboxylase, ribulose bis Examples thereof include phosphate carboxylase, phosphophenol pyruvate carboxylase, fructose bisphosphate aldolase, and phenylserine aldolase.
  • Examples of those that act on carbon-oxygen bonds include enolase, aconitase, carbonic anhydratase, tryptophan synthetase, threonine synthase, nitrile hydratase and the like.
  • Examples of the substance that acts on the carbon-nitrogen bond include aspartate ammonia lyase, arginosuccinate lyase, uroborufinogen I synthase, and the like.
  • cystathionine ⁇ -lyase examples include cystathionine ⁇ -lyase, cystathionine ⁇ -lyase, and methionine ⁇ -lyase.
  • Examples of phosphorus that act on oxygen binding include adenylate cyclase, guayule cyclase, and the like.
  • Examples of the isomerase include racemase and epimerase, cis-trans isomerase, intramolecular oxidoreductase, intramolecular transferase, intramolecular desorbing enzyme and the like.
  • racemase and isomerase examples include mutarotase, alanine racemase, serine racemase, amino acid racemase, and lactate racemase.
  • Examples of the cis-trans isomerase include maleate isomerase and linoleate isomerase.
  • Examples of the intramolecular oxidoreductase include phosphodanolose isomerase, xylose isomerase, mannose isomerase, and glucuronic acid isomerase.
  • intramolecular transferase examples include lysolecithin sacyl mutase, phosphodalysate phosphatase, lysine 2,3-amino mutase, methyl phospartate mutase and the like. It is.
  • Examples of the intramolecular elimination enzyme include flavonone lyase.
  • Synthetic enzymes include those that produce carbon-oxygen bonds, those that produce carbon-sulfur bonds, those that produce carbon-nitrogen bonds, those that produce carbon-carbon bonds, and those that produce phosphate esters. Etc.
  • Examples of those that generate carbon-oxygen bonds include tyrosyl-tRNA synthetase, leucyl 1-tRNA synthetase, methionyl 1-tRNA synthetase, lysyl 1-tRNA synthetase, glutaminorelation tRNA synthetase, and the like.
  • Examples of those that generate a carbon-sulfur bond include asinole- 1 CoA synthetase, acetyl-CoA synthetase, succinyl-CoA synthetase, and the like.
  • Examples of those that generate carbon-nitrogen bonds include glutamine synthetase, asparagine synthetase, oxidized nicotinamide adenine dinucleotide synthetase, glutathione synthetase, pantothenic acid synthetase, and guanosine monophosphate synthetase. , Urea carboxylase, dartame tocystine ligase and the like.
  • Examples of the carbon-carbon bond-forming agent include acetyl CoA carboxylase, pyruvate carboxylase, and propionyl CoA carboxylase.
  • Examples of those that produce phosphate esters include polydeoxyribonucleotide synthetase and polyribonucleotide synthetase.
  • the capture molecule in the present invention is a molecule that has binding affinity for both the molecular binding substance and the measurement target and can separate the measurement target and the contaminant contained in the sample. Means.
  • the molecule binding substance is a protein for binding a capture molecule such as protein A, protein G, protein L, or Fc receptor
  • a capture molecule that can bind to the protein for binding the capture molecule is used. Is preferred.
  • capture molecules in the present invention include antibodies and fragments thereof.
  • a molecule in which a ligand is bound to an antibody or antibody fragment examples include a molecule in which a ligand is bound to an antibody or antibody fragment, a complex containing an antibody or antibody fragment, a protein, a nucleic acid, a lipid, a sugar, a sugar chain, an aptamer (Nature, 1992, Vol.355, p. .85 0), including a site that captures the measurement target such as a polymer cage (Nature, 1993, Vol.361, P.645) made by molecular imprinting, and a site that binds to a molecular binding substance. Molecules.
  • the binding site to the molecular binding substance is selected according to the molecular binding substance. For example, when the molecular binding substance is protein A or protein G, Fc can be selected as the site, and when it is protein L, Fab can be selected.
  • the measurement target is an antigen specific to the antibody, a hapten, or a complex thereof.
  • the antigen complex refers to a substance containing other components such as an antibody or an enzyme in addition to the antigen.
  • a hapten complex refers to a substance containing other components in addition to a hapten, such as an antibody or an enzyme.
  • an antibody that is occupied by the antigen-antibody complex and is specific for another epitope is bound to a molecule binding substance.
  • the antibody as a capture molecule may be a whole molecule or a fragment as long as it binds to a molecule binding substance and a measurement object.
  • fragments Fab, F (ab), Fab ', F (ab')
  • Vc having one Fab and complete Fc Fc bound to a molecule that binds the substance to be measured, and combinations thereof.
  • Fc bound to a molecule that binds the substance to be measured can be prepared by a method using a known covalent bond or a known genetic engineering technique.
  • Examples of the capture molecule used in the present invention include a mouse antibody, a rat antibody, a rabbit antibody, a Hedge antibody, a chimeric antibody, a humanized antibody, a human antibody and the like without limitation.
  • the antibody is preferably a monoclonal antibody in that it can stably produce a homogeneous antibody, whether it is a polyclonal antibody or a monoclonal antibody.
  • Polyclonal and monoclonal antibodies can be produced by methods well known to those skilled in the art. If the molecular binding substance is an enzyme, no capture molecule is required.
  • the present invention is characterized in that a molecular binding substance and optionally a capture molecule are fixed to an aggregate region of a support having an aggregate region of two or more polymers having different stereoregularities on the surface.
  • the present invention also relates to a method for producing a carrier for measuring a measurement object in a sample.
  • the present invention also includes a step of fixing a molecular binding substance to an aggregate region of a support having an aggregate region of two or more polymers having different stereoregularities on the surface, and optionally a capture molecule for the molecular bond.
  • the present invention relates to a method for producing a carrier for measuring a measurement object in a sample, including a step of binding to a substance.
  • Various known methods can be used with no particular limitation on the method for immobilizing the molecular binding substance on the aggregate of two or more polymers having different stereoregularities.
  • a method in which a solution obtained by dissolving a molecular binding substance is brought into contact with the aggregate is preferable.
  • the contact method include a method of coating or spotting a solution on the aggregate, and examples of the coating method include dip coating, spin coating, gravure coating, spray coating, and the like.
  • the solution for dissolving the molecular binding substance an aqueous medium described later is suitable.
  • the concentration of the molecular binding substance contained in the solution may be any concentration, but it is desirable that the concentration is such that the molecular binding substance is bound to the aggregate in a saturated state. .
  • concentration of molecular binding substance contained in the dissolution solution typically 0. ⁇ ⁇ ⁇ / L or more, preferably 0. 1 ⁇ ⁇ / L or more, more preferably 0. 2 ⁇ ⁇ 0 1 / ⁇ more
  • the upper limit is provided a record as long as the material is dissolved with a suitable viscosity to solution, but generally lmmol / L or less, preferably 100 / i mol / L or less, further preferred properly is 50 im O l / L or less.
  • the temperature at the time of contact is preferably 0 ° C or higher, preferably 4 ° C or higher, more preferably 15 ° C or higher, and generally 70 ° C or lower. Preferably it is 50 ° C or less, more preferably 40 ° C or less.
  • the contact time can be appropriately selected depending on the temperature at the time of contact without any particular limitation. Generally, it is preferably 10 minutes or more, more preferably 30 minutes or more immediately after contact, and generally 48 hours or less, preferably 24 hours. Less than 2 hours, more preferably less than 2 hours.
  • the carrier of the present invention is a carrier in which the denaturation of the molecular binding substance fixed on the polymer aggregate is suppressed and storage stability is thereby improved, the carrier is in contact with the molecular binding substance. It is possible to leave it for a long time.
  • an enzyme when used as the molecular binding substance, various known methods may be used that are not particularly limited in the method of immobilizing the enzyme on an aggregate of two or more polymers having different stereoregularities. it can.
  • a solution obtained by dissolving an enzyme is contacted with the aggregate.
  • the touching method is preferred.
  • a contact method the above-mentioned contact method is mentioned, for example.
  • an aqueous medium described later is suitable.
  • the concentration of the enzyme contained in the lysate may be any concentration, but is preferably a concentration that achieves saturation of the enzyme on the aggregate.
  • the concentration of the enzyme contained in the lysate is generally 0.05 ⁇ mol / L or more, preferably 0.5 ⁇ mol / L or more, and more preferably 1 ⁇ mol / L or more. As long as it dissolves with an appropriate viscosity, there is no upper limit, but generally it is 100 ⁇ mol / L or less, preferably 10 ⁇ mol / L or less, more preferably 5 ⁇ mol / L or less.
  • the contact temperature is preferably a temperature at which the enzyme does not denature. Generally, it is 0 ° C or higher, preferably 4 ° C or higher, more preferably 15 ° C or higher, and generally 70 ° C or lower, preferably 50 ° C.
  • the contact time can be appropriately selected depending on the temperature at the time of contact without particular limitation, and is generally 10 minutes or more, more preferably 30 minutes or more immediately after contact, generally 48 hours or less, preferably 24 hours or less. More preferably, it is 2 hours or less. Since the carrier of the present invention is a carrier in which the denaturation of the enzyme immobilized on the polymer aggregate is suppressed and storage stability is thereby improved, the carrier is left in contact with the enzyme for a long time. Embodiments are possible.
  • a capture molecule when the above-described protein for binding a capture molecule is used as the substance for binding a molecule, a capture molecule can be further bound to the protein for binding a capture molecule, but there is no particular limitation on the method. Any known method can be used. For example, a method in which a solution in which a capture molecule is dissolved and a capture molecule-binding protein are brought into contact with each other can be mentioned.
  • the carrier of the present invention is a carrier for measuring a measurement object in a sample that binds to a capture molecule when the molecule binding substance is the above-described protein for binding a capture molecule.
  • a support having an aggregate region of two or more polymers having different stereoregularity, and a molecular binding substance fixed to the aggregate region of the support.
  • the capture molecule can be bound to the carrier via a molecule binding substance in some cases, and in the present specification, such a carrier is particularly referred to as a capture molecule-bound carrier.
  • a capture molecule-bound carrier is also a kind of a suitable carrier of the present invention.
  • the carrier of the present invention is a carrier for measuring a measurement object in a sample by an enzymatic reaction, or a continuous enzyme.
  • a carrier for enabling reaction comprising a support having an aggregate region of two or more polymers having different stereoregularities on the surface, and an enzyme immobilized on the aggregate region of the support.
  • the enzyme-immobilized carrier of the present invention can also be used, for example, in sensors, immunochromatography, bioreactors and the like.
  • the carrier and capture molecule-bound carrier of the present invention are preferably those subjected to a blocking treatment with a blocking agent.
  • the blocking agent preferably occupies a region not occupied by the molecular binding substance.
  • the blocking agent examples include substances that have an adsorptivity to water-absorbing base materials such as ushi serum albumin, casein, gelatin, skimminolek, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol, or block ace (Dainippon Pharmaceutical Co., Ltd.) ) And other commercially available blocking agents.
  • the blocking agent can be used by contacting a solution obtained by dissolving it in an appropriate solvent such as an aqueous solvent described later with the carrier of the present invention and the capture molecule-binding carrier.
  • the content of the blocking agent contained in the solution is generally 0.1% by weight or more, preferably 1% by weight or more, and there is no upper limit as long as it can be dissolved in the solvent, but preferably 10% by weight or less. is there.
  • Examples of the method for fixing the blocking agent include physical adsorption or chemical bonding which are generally used.
  • the contact conditions for immobilizing the aforementioned molecular binding substance may be used.
  • a carrier not bound to a capture molecule is prepared and stored in advance, and the capture molecule corresponding to the measurement object is bound immediately before the measurement to obtain a capture molecule-bound carrier.
  • An embodiment in which it is used for measurement is preferable.
  • a carrier subjected to blocking treatment with a blocking agent or a capture molecule-binding carrier is prepared and stored in advance and can be used immediately for measurement of a measurement object.
  • the carrier of the present invention in which the molecular binding substance is fixed to a support through the association of two or more polymers having different stereoregularities, is excellent in stability.
  • the carrier can be stored even in a dried state, and is suitable for use in the above embodiment.
  • the storage temperature in the dry state is preferably 2 ° C force 40 ° C, more preferably 4 ° C to 30 ° C.
  • the present invention is used for a carrier for measuring a measurement object in a sample by immobilizing a molecular binding substance in an aggregate region of two or more polymers having different stereoregularities of a support. It includes a polymer-attached support having two or more polymer aggregate regions having different stereoregularities on the surface.
  • the polymer-attached support is used for measurement by immobilizing a substance for molecular binding before measurement and further binding a capture molecule.
  • the molecule binding substance is, for example, protein A (one of the capture molecule binding proteins)
  • protein A is deposited on the polymer aggregate film containing the polymetatalate stereocomplex. Can be fixed directly. As a result, it is possible to easily prepare the carrier. Further, by providing the polymer aggregate film, it is possible to suppress deterioration of the performance of the carrier after protein A fixation over time, and storage stability is improved. Compared to the conventional immobilization method, it is considered that the denaturation of protein A is suppressed by using the direct interaction between the polymer-associated membrane and protein A. Furthermore, it is possible to increase the amount of measurement object binding per unit area, thereby improving measurement sensitivity.
  • protein A does not grow in three dimensions (SK mode) but layered (FM mode) on the polymer film, and is used for antibody binding. It is also thought that the area increases and the antibody binds efficiently.
  • the binding surface of protein A to the antibody can be presented to the antibody so that the antibody has an effect of binding, which makes it easier for the antibody to bind. Is also possible.
  • the enzyme when the molecular binding substance is an enzyme, the enzyme can be directly immobilized on a polymer aggregate film containing a stereocomplex of polymetatalylate. As a result, the carrier can be easily prepared.
  • the polymer aggregate membrane by providing the polymer aggregate membrane, it is possible to suppress the deterioration of the performance of the carrier after immobilization of the enzyme over time, and the storage stability is improved. Compared to the conventional immobilization method, it is considered that the denaturation of the enzyme is suppressed by using the direct interaction between the polymer-associated membrane and the enzyme. It is.
  • the enzyme reaction efficiency per unit area can be increased, resulting in improved measurement sensitivity or product production efficiency.
  • the enzyme grows in layers (FM mode) instead of three-dimensional growth (SK mode) on the polymer film, and is used for binding of the substrate and the enzyme. It is also thought that the area increases and the substrate comes into efficient contact with the enzyme.
  • the binding surface with the substrate that the enzyme has can be presented to the substrate so that the effect of binding the substrate can be obtained, which also makes it easier for the substrate to bind. It is done.
  • the present invention also relates to a method for measuring an object to be measured in a sample using the carrier of the present invention.
  • the sample used in the measurement method of the present invention is not particularly limited as long as it is a sample that enables the measurement method of the present invention, for example, a biological fluid derived from a human or non-human animal (blood fluid, serum, plasma, Urine, feces, sputum, saliva, intraperitoneal fluid, etc.), plants * fluids separated and extracted from microorganisms, fluids separated and extracted from food 'food raw materials, fluids generated from the manufacturing process of chemicals, rivers' seawater, Examples include liquids that are separated and extracted from the environment such as air and soil, and liquids that are artificially added to a buffer solution or the like so that the measurement target has a known concentration or a certain concentration.
  • a biological fluid derived from a human or non-human animal blood fluid, serum, plasma, Urine, feces, sputum, saliva, intraperitoneal fluid, etc.
  • plants * fluids separated and extracted from microorganisms fluids separated and extracted from food 'food raw materials
  • the measurement method of the present invention comprises a step of bringing the aforementioned carrier into contact with the measurement object in a reaction solution and reacting the measurement object with an enzyme immobilized on the carrier, and increasing or decreasing by the reaction. Measuring a substance to be measured.
  • the measurement method of the present invention comprises a step of bringing the aforementioned carrier and a capture molecule into contact with each other in a reaction solution to form a capture molecule-bound carrier, and bringing the capture molecule-bound carrier and a measurement object into contact with each other.
  • the measurement method of the present invention includes a step of bringing a capture molecule and a measurement object into contact with each other in a reaction solution to form a measurement object-bound capture molecule, and contacting the carrier and the measurement object-bound capture molecule. And a step of binding the measurement object to a carrier and a step of measuring the measurement object bound to the carrier.
  • the method of the present invention comprises a step of bringing a capture molecule-binding carrier and a measurement object into contact with each other in a reaction solution, and binding the measurement object to the support via the capture molecule.
  • a step of measuring a measurement object comprises a step of bringing a capture molecule-binding carrier and a measurement object into contact with each other in a reaction solution, and binding the measurement object to the support via the capture molecule.
  • the reaction solution used in the measurement method of the present invention is not particularly limited as long as the carrier and the measurement object, the carrier and the capture molecule, or the solvent capable of reacting the capture molecule-bound carrier and the measurement object are used.
  • an aqueous medium an organic solvent, a supercritical fluid and the like can be mentioned, but an aqueous medium is preferable.
  • a force buffer solution such as deionized water, distilled water, and a buffer solution is preferable.
  • the buffer used in the buffer solution is not particularly limited as long as it has a buffering capacity, but, for example, lactic acid buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer having a pH of 1 to 11 , Phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, tris (hydroxymethyl) aminomethane buffer, imidazole buffer, malate buffer, oxalate buffer, glycine Examples thereof include a buffer, a borate buffer, a carbonate buffer, and a Good buffer.
  • Good buffering agents include, for example, 2_morpholinoethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N- (2-acetamido) iminoniacetic acid (ADA) Piperazine N, N 'Bis (2-ethanesulfonic acid) (PIPES), N- (2-acetamido) 2 Aminoethanesulfonic acid (ACES), 3-morpholino 1-hydroxypropanesulfonic acid (M ⁇ PS ⁇ ), N, N Bis (2-hydroxyethyl) 2-aminoethanesulfonic acid (BES), 3-morpholinopropanesulfonic acid (MOPS), N- [tris (hydroxymethyl) methyl] 2 aminoethanesulfone Acid (TES), 2- [4- (2 Hydroxyethyl) 1-piperadul] ethanesulfonic acid (HEPES), 3- [N, N Bis (2-hydroxyxe
  • Examples of the organic solvent include ethanol, methanol, acetonitrile, dimethylformamide, dioxane, hexane, benzene, isooctane and the like.
  • Examples of the supercritical fluid include supercritical carbon dioxide, supercritical water, supercritical ethanol, and supercritical methanol.
  • the reaction temperature is not particularly limited as long as a specific reaction can be performed, but is generally 0 ° C or higher, preferably 4 ° C or higher, more preferably 15 ° C or higher. Generally, it is 70 ° C or lower, preferably 50 ° C or lower, more preferably 40 ° C or lower.
  • the reaction time can be appropriately selected depending on the reaction temperature without any particular limitation. Generally, immediately after the reaction, it is preferably 1 minute or more, more preferably 5 minutes or more, and generally 24 hours or less, preferably 4 hours or less. More preferably, it is 2 hours or less.
  • the measurement method of the present invention can be used for both quantitative and qualitative applications.
  • the change in physical properties to be measured can be measured quantitatively and converted to the amount of the combined measurement object.
  • the carrier for example, by immobilizing different capture molecules in a plurality of capture molecule binding regions on the carrier, quantitatively measuring the amount of change in physical properties in each region, and converting it to the amount of bound measurement object, It is also possible to quantify multiple measurement objects at once.
  • the molecule binding substance is a capture molecule binding protein or an enzyme
  • the measurement method of the measurement object and the measurement kit will be described in detail for each case.
  • Chi is a hidden foot chi ffl protein
  • the measurement target can be measured by detecting a change in physical properties caused by the binding between the measurement target and the carrier.
  • the measuring method is appropriately selected according to the physical properties.
  • the physical properties include spectroscopic characteristics, luminescence and fluorescence, radiation emission, physical shape, dielectric constant, optical properties, weight, and the like. Any known measuring means is selected according to the change in physical properties to be measured.
  • the measurement object is labeled with an antibody to which a labeling substance is bound, or a complex containing the labeling substance and the antibody (hereinafter collectively referred to as an antibody labeling complex).
  • the label can be appropriately selected from enzymes, fluorescent substances, radioisotopes, colloids and the like.
  • carrier aggregation is measured. Suitable supports for this embodiment include fine particles such as latex or gold colloid.
  • the label is an enzyme (generally called an ELISA method)
  • a dye can be produced by an enzymatic reaction and detected by a known spectroscopic device.
  • a known chemiluminescence detector can be used.
  • the label is a fluorescent substance
  • a known fluorescence measuring device can be used.
  • the label is a radioisotope (generally called a radioimmunoassay method)
  • a known radiation detection apparatus can be used.
  • the label is a colloid, it may be observed directly with a scanning probe microscope such as an atomic force microscope or a scanning tunneling microscope or an electron microscope.
  • a measurement object labeled with a colored substance such as a gold colloid
  • it can also be detected spectroscopically.
  • spectroscopic techniques such as change in absorbance and light dispersion are used.
  • the crystal oscillation method measures changes in vibration frequency
  • the surface plasmon resonance method (SPR) measures the change in reflection angle caused by the change in dielectric constant
  • the micro differential thermal measurement method measures heat.
  • a measurement object that is not labeled it may be observed directly with a high-resolution electron microscope, a scanning probe microscope, a scanning near-field microscope, or the like.
  • an arbitrary measuring device according to the labeling mode specifically a spectroscopic device, a fluorescence test, An extraction device, a chemiluminescence detection device, a radiation detection device, or the like can be used.
  • the spectroscopic methods used include infrared absorption, ultraviolet visible absorption, Raman scattering and the like.
  • the carrier of the present invention By using the carrier of the present invention, it is also possible to simultaneously measure a plurality of measurement objects in a measurement sample.
  • the carrier of the present invention in combination, that is, by immobilizing different types of capture molecules depending on the measurement object, a plurality of types of measurement objects can be measured simultaneously.
  • This simultaneous multi-item measurement method speeds up the examination and diagnosis.
  • a plurality of capture molecule binding regions may be provided on one carrier, and different capture molecules may be bound to each region.
  • a large number of samples can be measured at once, and screening can be accelerated.
  • a capture molecule in advance on a carrier such as 96 or 384 plate, bead or chip.
  • a carrier such as 96 or 384 plate, bead or chip.
  • a means is required to know which analyte is being captured for the capture molecule binding region force S.
  • a means for associating the beads with the measurement object is required.
  • it is possible to identify beads by irradiating a laser beam and classifying in detail the signals emitted from the fluorescent dye held by the beads. It is.
  • the bead can be identified by detecting the barcode attached to the surface of the capture molecule binding region by the most suitable method.
  • Specific measurement apparatuses capable of identifying the capture molecule binding region include the following.
  • a plate it is a plate reader equipped with a detector corresponding to a changing physical property.
  • a measuring device including an image analysis device equipped with a detector corresponding to a changing physical property (eg, Perkin Elmer).
  • an image analysis device equipped with a detector corresponding to a changing physical property (eg, Perkin Elmer).
  • beads equipped with a detector that reads the identification symbol attached to the beads and a detector that responds to changing physical properties
  • a measuring device eg, Luminex 100, Luminex
  • the present invention also relates to a measurement kit comprising the carrier described above.
  • a measurement kit comprising the carrier described above.
  • any known element can be selected according to the measurement technique.
  • Examples of the ELISA method include enzyme-labeled antibody, enzyme-labeled antibody diluent, enzyme reaction substrate solution, enzyme reaction stop solution, and washing solution. Furthermore, blocking agents, standard substances, measurement sample diluents, and the like can be optionally added.
  • fluorescence immunoassay examples include fluorescently labeled antibodies, fluorescently labeled antibody dilutions, and washing solutions. Furthermore, blocking agents, standard substances, measurement sample diluents, and the like can be arbitrarily added.
  • radioimmunoassay method examples include radiolabeled antibodies, radiolabeled antibody diluents, washing solutions, and the like. Furthermore, it is possible to add a blocking agent, standard substance, measurement sample diluent, etc., arbitrarily.
  • the measurement kit includes a test strip in which a membrane including an antibody binding region is used as a carrier and a sample pad, a conjugate pad, an absorption pad and the like are arranged in a housing.
  • Constituent elements other than the test strip in the measurement kit include labeled antibodies having a label arbitrarily selected from enzymes, fluorescent substances, radioactive substances, gold colloids, etc., labeled antibody diluents, washing solutions and the like.
  • blocking agents, standard substances, measurement sample diluents, and the like can be optionally added.
  • the measurement object of the present invention includes human serum albumin (HSA), ushi serum albumin (B
  • Fig. 1 shows one embodiment of the formation of the carrier of the present invention and the capture molecule-binding carrier when the capture molecule-binding protein is used as the molecule-binding substance, and the reaction between the carrier and the measurement object. .
  • the measurement object can be measured by measuring the amount of reaction product that increases due to the reaction between the measurement object and the enzyme, or the amount of reaction substrate that decreases. .
  • the reaction product or the reaction substrate can be directly measured, or it can be converted into an appropriate substance and measured.
  • the measurement method and conversion method are selected as appropriate.
  • the measurement includes spectroscopic measurement, luminescence and fluorescence measurement, dielectric constant measurement, and the like, and any known measurement means is selected according to the substance to be measured.
  • the reaction product is a dye or a substance that absorbs light of a specific wavelength.
  • a known chemiluminescence detector is used.
  • the reaction product is a fluorescent material, a known fluorescence measuring device is used. Can be used.
  • the embodiment of the sensor equipped with electrodes is more preferable.
  • the electrode include a hydrogen peroxide electrode, a dissolved oxygen electrode, a conductivity electrode, an ion electrode, a pH electrode, and a redox electrode.
  • the electrode can be properly used depending on the type of reaction product. For example, if the reaction product is hydrogen peroxide, the hydrogen peroxide electrode, if it is an electron or a charged substance or ion, the conductivity electrode, ion electrode or pH electrode, dissolved if the reaction involves oxygen consumption An oxygen electrode or the like can be used as long as the reaction causes a change in the redox potential.
  • a sensor provided with an electrode is suitable for a device that is easy to carry because it can form a small and simple device.
  • Two or more kinds of enzymes may be immobilized on the carrier (enzyme-immobilized carrier) of the present invention.
  • a detectable substance die, light, fluorescence, etc.
  • all or part of the two or more types of enzymes are used in the aggregate region of the support. Fixing force S
  • the carrier of the present invention enzyme-immobilized carrier
  • the carrier of the present invention in combination, that is, by immobilizing different types of enzymes depending on the measurement target, multiple types of measurement target can be measured simultaneously using one sample. Can do.
  • This simultaneous multi-item measurement method speeds up the examination and diagnosis.
  • a plurality of enzyme binding regions may be provided on one carrier, and different enzymes may be bound to each region. By binding a plurality of types of enzymes in one carrier, a large number of samples can be measured at a time, and screening can be accelerated.
  • Specific measurement apparatuses capable of identifying the enzyme binding region include the following.
  • a plate it is a plate reader equipped with a detector corresponding to the amount of substance that changes
  • a chip it includes a measuring device including an image analysis device equipped with a detector according to the amount of substance that changes (eg: Perkin Elma company scan array series).
  • the carrier (enzyme-immobilized carrier) of the present invention is also suitable when a plurality of samples are continuously measured.
  • this aspect is possible by putting a plurality of samples into another container and sequentially contacting the carrier with the samples in the container.
  • the sample can be flowed so that a plurality of samples do not cross each other in the flow channel.
  • a diagnostic bioreactor using the carrier (enzyme-immobilized carrier) of the present invention is suitable for measuring a plurality of samples continuously.
  • the diagnostic enzyme is not disposable but is fixed in the reactor (reactor) for repeated use.
  • a porous alkylamine glass (particle size: 125 to 177 ⁇ m, pore size: 50 nm) is coated as a support with the polymer of the present invention, and this is coated with glucose oxidase or cholesterol. Fix oxidase etc.
  • a flow type is provided by providing a sample inlet on one side of this diagnostic bioreactor, an outlet for the reaction product on the other side, and a sensor (detector of the detector) for measuring the reaction product at the outlet.
  • a diagnostic bioreactor can be made.
  • the flow-type diagnostic bioreactor can send a sample in one direction without the need to put a sensor into the sample for each specimen, so that multi-directional diffusion of substrates and reaction products is unlikely to occur. This is particularly important when two or more enzyme reactions are carried out continuously, by connecting the reactors of different enzymes in a straight line, so that the first reaction is incomplete. The possibility of the reaction taking place can be kept to a minimum.
  • the change in the amount of substance caused by the measurement object in the sample previously contacted may affect the change in the amount of substance caused by the measurement object in the sample contacted later.
  • the present invention also relates to a measurement kit including a carrier using the aforementioned enzyme.
  • Arbitrary well-known elements can be selected as components other than the carrier in the measurement kit. That is, an enzyme reaction stop solution, a blocking agent, a standard substance, a measurement sample diluent, a cleaning solution, and the like can be arbitrarily added.
  • the measurement kit includes a test strip in which a membrane including an enzyme immobilization region is used as a carrier and a sample pad, a conjugate pad, an absorption pad and the like are arranged in a housing.
  • a test strip in which a membrane including an enzyme immobilization region is used as a carrier and a sample pad, a conjugate pad, an absorption pad and the like are arranged in a housing.
  • an enzyme reaction stop solution, blocking agent, standard substance, measurement sample diluent, washing solution, etc. may be added arbitrarily.
  • the measurement object that can be measured by the carrier of the present invention includes LDL cholesterol, HDL cholesterol, total cholesterol, free cholesterol, triglyceride, phospholipid, free fatty acid, lipid peroxide, creatinine.
  • the carrier of the present invention can also be applied to the production of substances.
  • the reaction solution containing the reaction product is immediately separated from the enzyme, thereby reducing the inhibition of the enzyme reaction by the reaction product.
  • Embodiments made possible by the present invention also include the continuous removal of the reaction product produced by the enzyme. Further, the convenience is further improved by combining the modes in which the substrate can be continuously contacted with the carrier. The mode in which the substrate is continuously contacted and the reaction product can be continuously taken out makes it possible to simplify the apparatus for continuously producing the reaction product.
  • Production of a substance using the carrier of the present invention can be performed, for example, on the surface of the carrier. Can be carried out by continuously flowing a reaction solution containing the substrate.
  • the carrier can also function as a production means for continuously removing the reaction product and a means for bringing the substrate into continuous contact with the carrier.
  • the carrier of the present invention is formed in a part of the channel or when the carrier is present in the channel, the substrate and the carrier can be brought into contact by flowing a reaction solution containing the substrate through the channel. .
  • Examples of the case where the carrier of the present invention forms a part of the flow path include micro-TAS (Micro-Total Analysis System), MEMS (Micro-Electro Mechanical System) or lab-on-a-chip (Lab— On-a-chip) is a mode in which a carrier is installed in a microchannel.
  • An example of the case where a carrier is present in the flow path is an embodiment in which beads are used as a support and packed in a column that is a flow path, such as an enzyme reactor or a bioreactor.
  • Production of a substance using a carrier can also be performed by introducing the carrier of the present invention into a reaction solution containing a substrate.
  • the reaction solution and the carrier are separated by putting a carrier such as a bead into the container containing the reaction solution, allowing the enzyme reaction to occur, and then capturing the carrier with a membrane having a hole of a size that does not pass through the carrier. Means are preferred.
  • Examples of the apparatus for continuously producing the reaction product include an enzyme reaction column, an enzyme reactor, and a bioreactor.
  • Examples of the apparatus for continuously producing the reaction product include a continuous stirring reactor, a packed bed reactor, a fluidized bed reactor, a membrane reactor, a hollow fiber reactor, and a rotating reactor. Examples include disk reactors, magnetic field reactors, and two-phase reactors.
  • reaction product that can be produced according to the present invention is not limited as long as it can be produced by an enzymatic reaction.
  • alcohols such as ethanol, polyphenols, trans-4-hydroxyproline, glycopeptides, sugars, etc.
  • Lipids monosaccharides, polysaccharides such as cyclodextrins, oligosaccharides such as trehalose, amino acids such as L-aspartic acid, L-alanine and L-tryptophan, oligopeptides such as dipeptides and aspartame, lipids, nucleic acids, Sweeteners such as isomerized sugar, umami substances such as inosinic acid, food additives such as diacinoreglycerol, food raw materials such as lactose-decomposed milk and casein decomposition products, raw materials for cosmetics, synthetic polymers such as acrylic amide Raw materials for antibiotics such as 6-aminopenicillane and 7-aminocephalosporin, raw materials for steroid hormones such as 1,4_androstagene and 4_androstene, human insulin, human growth hormone, etc.
  • Polypeptide pharmaceuticals L-dopa, (6S) -tetrahydrofolic acid and other pharmaceuticals
  • PMMA Polymer Source. Inc. or Polyscience, Inc.
  • Mn represents the number average molecular weight
  • Mw represents the weight average molecular weight
  • mm / mr ⁇ is a triplet symbol representing stereoregularity
  • mm is the isotacticity
  • rr is (It-PMMA stands for isotactic PMMA, st-PMMA stands for syndiotactic PMMA, and at-PMMA stands for atactic PMMA.)
  • Protein A manufactured by Cosmo Bio. The protein A solution was prepared as follows.
  • Protein A (Mw 42,000) stored at 4 ° C was returned to room temperature and dissolved in 0.02 wt% sodium azide (NaN) / PBS (phosphate buffered saline) solution (2.38 mL). 20 ⁇ mol / L
  • Mouth Tin A solution was prepared and allowed to stand at 4 ° C for 2 hours.
  • the protein A solution was aliquoted at 150 a L, spun down, frozen in liquid nitrogen, and stored at -20 ° C. Before use, it was thawed and diluted to 1 ⁇ mol / L with PBS.
  • HSA Anti-human serum albumin
  • the frozen antibody solution was quickly thawed at 37 ° C, diluted with PBS to 1 ⁇ mol / L, and stored at 4 ° C. At the time of use, 1 mL was sampled into a polypropylene Sampnore tube.
  • PBS Ca, Mg free
  • BSA urine serum albumin
  • HSA is an antigen specific for anti-human HSA antibody
  • BSA is a non-specific antigen used as a control.
  • Both HSA and BSA solutions were prepared as follows. First, albumin is allowed to stand at room temperature for 30 minutes in a desiccator, and a predetermined amount is weighed. When completely dissolved, prepare an amount of PBS that will give an albumin concentration of 10 ⁇ mol / L, and continue until 4 days at 4 ° C. Allowed to stand to dissolve completely. Further diluted with PBS to 1 ⁇ mol / L. These solutions were used within one week of preparation.
  • ⁇ -galatatosidase [ ⁇ _D_galactosidase; derived from E. coli, 5-30 unit ZmL; manufactured by Wako Pure Chemical Industries, Ltd.].
  • PNPG p-Nitrophenyl extra viranoside
  • the polymer membrane / protein A comprising the support / stereo complex was prepared as follows.
  • An AT-cut quartz crystal (QCM) chip with a fundamental frequency of 9 MHz is used as a support, and a carrier having a polymer film of a stereo complex of it-PMMA and st-PMMA on the support is as follows. It produced as follows. In addition, a gold thin film was formed on the QCM chip, and a polymer film was formed on the gold thin film.
  • QCM AT-cut quartz crystal
  • This QCM chip was immersed in a 1.7 mg / mL it-PMMA solution (solvent: acetonitrile) for 5 minutes at 25 ° C, pulled up, washed with acetonitrile (special grade), and then washed with ultrapure water. Dry with nitrogen gas. Then, add 25 mg to 1.7 mg / mL st-PMMA solution (solvent: acetonitrile). It was immersed in C for 5 minutes, pulled up, washed with acetonitrile (special grade), then washed with ultrapure water, and dried with nitrogen gas. This operation is one cycle and this cycle is repeated for a total of 14 times. went. In this way, a polymer film was formed.
  • Protein A was immobilized on the polymer film by spin coating.
  • the support on which the above polymer film was formed was immersed in a 1 ⁇ mol / L protein A solution at 37 ° C. for 60 minutes, washed with ultrapure water, and dried to prepare a carrier.
  • Comparative Example 1 Support / stereocomplex polymer membrane (without protein A) A measurement carrier was prepared in the same manner as in Example 1 except that protein A was not fixed.
  • a QCM chip similar to that in Example 1 is used as a support, and it_PMMA alone is provided on each support.
  • a carrier for measurement was prepared using a support on which a polymer film composed of (Comparative Example 2), st_PMMA alone (Comparative Example 3), and at-PMMA (Comparative Example 4) was formed.
  • the polymer film was prepared by mounting one drop of the above it_PMMA, st-PMMA, and at_PMMA solution (solvent: black mouth form) on a support with a Pasteur pipette and spin coating (2000 rpm,
  • the QCM chip was washed with Piranha solution and dried to use as a measurement carrier.
  • Protein A was further immobilized on the measurement carriers of Comparative Examples 2-4.
  • the polymer films are respectively it-PMMA alone (Comparative Example 6), st_PMMA alone (Comparative Example 7), and at-PMMA (Comparative Example 8).
  • Protein A was immobilized in the same manner as in Example 1. To determine the amount of protein A immobilized, the frequency was measured before and after protein A immobilization.
  • Comparative Example 9 Support / Protein A Protein A was immobilized on the carrier for measurement of Comparative Example 5. Protein A was immobilized in the same manner as in Example 1 and dried to prepare a carrier. To determine the amount of protein A immobilized, the frequency was measured before and after protein A immobilization.
  • Table 1 shows the structures of the carriers prepared in Example 1 and Comparative Examples:! -9.
  • Comparative Example 7 Support Zst-PMMA single membrane / protein A
  • Example 2 shows the amount of fixed protein A (protein A fixed amount) as the amount of change in frequency.
  • the carrier prepared in Example 1 was immersed in a 1 ⁇ mol / L anti-human HSA antibody solution at 37 ° C. for 60 minutes to bind the antibody. Next, remove from the solution, wash with ultrapure water at 37 ° C, immerse in PBS solution for 1 hour without drying, then wash with ultrapure water at 37 ° C and dry to produce anti-human HSA antibody-binding carrier did.
  • the antibody was bound to the measurement carrier prepared in Example 1 (Protein A fixed treatment), Comparative Examples:! To 3 and 5 (No Protein A fixed treatment), and the binding amount was evaluated. Furthermore, antigens were further bound and the amount of binding was evaluated, and the effect of protein A fixation on the antibody and antigen binding amount was tested.
  • the amount of antibody binding was evaluated as follows.
  • Table 3 shows the results of antibody binding and antigen binding.
  • the amount of coupling is expressed in terms of frequency change (Hz).
  • the average number of moles of HSA bound per mole of antibody (denoted as HSA / antibody in the table) was calculated from the amount of antibody bound, the amount of HSA bound, and the molecular weight of each.
  • Example 1 immobilizes protein A, and Comparative Examples 1 to 3 and 5 do not immobilize protein A
  • HSA / antibody ratio is the molar ratio calculated from the change in frequency.
  • fixing the protein A not only increases the amount of antibody binding, but also increases the specific antigen binding ratio per antibody (HSA / antibody). This result suggests that the denaturation of the antibody can be suppressed by intervening protein A, and / or that the binding state of the antibody is controlled to promote the antigen binding.
  • Antibodies were bound using the measurement carriers prepared in the measurement carriers of Example 1 and Comparative Examples 6 to 9 (all of which protein A is immobilized), and the amount of antibody binding was evaluated. Furthermore, antigens were further bound to evaluate the amount of antigen binding, and the effects of the polymer membrane were compared. Table 4 shows the results of antibody binding and antigen binding. The amount of coupling is expressed in terms of frequency change (Hz). The average number of moles of HSA bound per mole of antibody (denoted as HSA / antibody in the table) was calculated from the amount of antibody bound, the amount of HSA bound, and the molecular weight of each.
  • Hz frequency change
  • Protein A is immobilized on any measurement carrier.
  • HSA / antibody ratio is the molar ratio calculated from the change in frequency.
  • it-PMMA single film Comparative Example 6
  • st-PMMA single film Comparative Example 7
  • at-PMMA Comparative Example 8
  • the measurement carrier of the present invention exhibits excellent performance in that a very small amount of measurement object can be measured efficiently.
  • Example 1 The measurement carriers of Example 1, Comparative Example 6, and Comparative Example 9 were stored for a certain period after preparation, and the antibody binding amount and the antigen binding amount were measured in the same manner as in Test Example 2 to change the performance over time. Examined.
  • the storage condition was that the sample was stored in a silica gel container protected from light at room temperature, and the measurement was performed 1, 3, 5, and 10 days after the start of storage. The results after 10 days are shown in Table 5.
  • Protein A is immobilized on all measurement carriers.
  • each measurement carrier is anti-human HSA anti-antibody of ⁇ ⁇ ⁇ / L at 37 ° C. After immersing in the body solution for 60 minutes to bind the antibody, it was taken out from the solution and washed with ultrapure water at 37 ° C. This measurement carrier was immersed in a PBS solution for 1 hour without drying, washed with ultrapure water at 37 ° C., and dried. Thereafter, the frequency was measured, and the amount of antibody binding was evaluated based on the difference from the initial value.
  • Antigen binding was performed as follows. The measurement carrier was immersed in a 1 ⁇ mol / L antibody solution at 37 ° C. for 60 minutes to bind the antibody, and then removed from the solution and washed with ultrapure water at 37 ° C. Without drying this measurement carrier, immerse it in a sample solution containing HSA antigen adjusted to a concentration of 0.1, 0.5, 1.0, 5.0 ⁇ mol / L at 37 ° C for 1 hour. After binding the HSA antigen, it was taken out from the solution, washed with ultrapure water at 37 ° C., and dried. A product which was washed with ultrapure water at 37 ° C. without binding HSA antigen and dried was also prepared.
  • Fig. 3 shows a calibration curve showing the relationship between the amount of HSA antigen binding and HSA antigen concentration. The amount of coupling is expressed in terms of frequency change (Hz).
  • a calibration curve showing the relationship between the HSA antigen binding amount and the HSA antigen concentration was prepared in the same manner as in Example 3 using the carrier of Comparative Example 6 instead of the carrier of Example 1.
  • the HSA antigen concentrations were 0.5, 1.0, and 5. ⁇ mol / L. The results are shown in Figure 3.
  • a calibration curve representing the relationship between the HSA antigen binding amount and the HSA antigen concentration was prepared in the same manner as in Example 3 using the carrier of Comparative Example 9 instead of the carrier of Example 1.
  • the HSA antigen concentrations were 0.5, 1.0, and 5. ⁇ mol / L. The results are shown in Figure 3.
  • the carrier using the stereocomplex membrane is the same concentration of HSA as compared to the case where the it_PMMA single membrane (Comparative Example 6) was used and the case where the polymer membrane was not used (Comparative Example 9).
  • the amount of HSA antigen binding increases with respect to the antigen concentration. This indicates that a carrier using a stereocomplex membrane is superior as a carrier for quantification of HSA antigen.
  • ⁇ -galatatosidase was dissolved in 50 mmol / L phosphate buffer containing lmmol / L magnesium chloride so that the concentration was 0 ⁇ 0025 to 1 ⁇ mol / L to prepare a j3_galactosidase solution.
  • a QCM chip formed with a polymer film composed of it_PMMA alone in Comparative Example 1 was immersed in each ⁇ -galatatosidase solution at each concentration for 60 minutes at room temperature, washed 3 times with 50 mmol / L phosphate buffer, and dried.
  • a ⁇ -galatatosidase-binding carrier was prepared.
  • ⁇ -Galatatosidase was dissolved in a 50 mmol / L phosphate buffer containing lmmol / L magnesium chloride so that the concentration was 0.0025 to 1 ⁇ mol / L to prepare a _galactosidase solution.
  • a QCM chip formed with a polymer film composed of it-PMMA alone in Comparative Example 2 was immersed for 60 minutes at room temperature, washed with 50 mmol / L phosphate buffer three times, It was dried to prepare a ⁇ -galatatosidase-binding carrier.
  • AF is the frequency change amount
  • Am is the amount of ⁇ -galactosidase binding
  • ⁇ is the electrode area
  • P is the density of the crystal
  • ⁇ is the shear stress of the crystal.
  • the amount of -galactosidase binding per cm 2 was calculated by dividing the amount of ⁇ -galatatosidase binding ( ⁇ m, unit ng) bound to the quartz crystal electrode by the area of the electrode.
  • Table 6 summarizes the amount of change in frequency and the amount of ⁇ -galliary sidase binding per lcm 2 for each concentration of ⁇ -galliary sidase solution used for immersion.
  • Fig. 4 shows the results of plotting data with the concentration of ⁇ -galactosidase solution on the horizontal axis and the amount of ⁇ -galatatosidase binding per lcm 2 on the vertical axis.
  • Example 4 the amount of ⁇ -galatatosidase bound to the QCM chip increased depending on the concentration of ⁇ -galatatosidase solution S, and the concentration of ⁇ -galatatosidase solution was 0.25 ⁇ ⁇ ⁇ Above 1 / ⁇ , the / 3 -extra-galliary sidase binding was saturated and did not increase any more. This result shows that in order to saturate the amount of ⁇ -galatatosidase bound to the QCM chip, it should be immersed in a / 3 -galatatosidase solution with a concentration of 0.25 ⁇ mol / L or higher.
  • A is the amount of ⁇ -galatatosidase binding
  • ⁇ is the maximum binding of ⁇ -galatatosidase
  • K is the apparent binding constant
  • [ ⁇ -Gal] is the concentration of ⁇ -galatatosidase.
  • Example 4 using a polymer film of stereocomplex, the maximum binding amount of / 3-galactosidase is large compared to Comparative Example 12 using a polymer film of it-PMMA alone. It was slight. The maximum binding amount of ⁇ -galactosidase of Example 4 was only 1.3 times that of Comparative Example 12.
  • Comparative Example 1 The j3_galactosidase solution was removed, washed 3 times with 50 mmol / L phosphate buffer, and dried to prepare a measurement carrier. This was designated as Comparative Example 1 3. Comparative Example 14 was prepared by immobilizing ⁇ -galatatosidase on the well of a NUNKU 96-well maxisorp plate in which a polymer film was not formed in the same manner as in Example 5.
  • PNPG ⁇ -Nitrophenyl digalatatoviranoside
  • FIG. 5 shows a plot of absorbance at a wavelength of 405 nm with respect to the time after adding PNPG for each concentration of 0.125, 0.25, 0.5, and ⁇ ol / L of the j3_galactosidase solution used for fixation. It was shown to.
  • Example 5 In any of the cases of Example 5 and Comparative Examples 13 and 14, the enzymatic reaction proceeds with time, but the activity value is the highest in the case of the polymer film comprising the stereocomplex of Example 5. Became high. Divide the absorbance change from 5 to 20 minutes in Figure 5 by the amount of ⁇ -galatatosidase immobilized on the wells of the 96-well maxisorp plate at each concentration of -galactosidase to give unit dose (mg) and unit time. The ⁇ -galactosidase activity per minute was determined and summarized in Table 8.
  • the amount of 13-galatatosidase bound to the well of the 96-well maxisorp plate was calculated by calculating the contact area of the ⁇ -galatatosidase solution in the tool, and the area value was calculated as / 3 -galatatosidase per lcm 2 in Table 6. It can be calculated by multiplying the amount of binding.
  • Example 5 and Comparative Examples 13 and 14 are stored for a certain period of time after preparation, and ⁇ -galatatosidase activity is measured by the same method as in Test Example 6 to determine the performance. The change with time was examined.
  • the storage condition is that the sample should be stored in a container with silica gel protected from light at room temperature. went.
  • Fig. 6 shows a plot of absorbance at a wavelength of 405 nm versus time after the addition of PNPG for the concentration power of Si-a-mol / L of the ⁇ -galatatosidase solution used for immobilization.
  • Example 5 In any case of Example 5 and Comparative Examples 13 and 14, the activity value increased with time, but the activity value was highest in the polymer film comprising the stereocomplex of Example 5.
  • the amount of j3-galatatosidase activity per unit amount (mg) and unit time (min) was determined from the change in absorbance over 5 to 20 minutes in Fig. 6 and summarized in Table 9.
  • Example 5 In all cases of Example 5 and Comparative Examples 13 and 14, the force that was found to be lower than the pre-storage activity by storage for 7 days In the case of Comparative Example 13, the activity before storage When the value is 100%, the activity value after 7 days is 8.5%, whereas in Example 5, the activity value after 7 days is 14.6 when the activity value before storage is 100%. %, And the rate of decrease is smaller in Example 5. This force It is considered that the activity of ⁇ -galatatosidase immobilized on the stereocomplex of Example 5 is maintained.
  • the measurement carrier of the present invention an aggregate of two or more polymers having different stereoregularities is formed on a support, and therefore, the protein carrier immobilized on the polymer aggregate is Deformation is suppressed and excellent storage stability is exhibited. Furthermore, since the amount of antibody binding and antigen binding per unit area increases, it is possible to improve measurement accuracy and sensitivity, and to quickly measure a small amount of sample. Therefore, according to the present invention, there are provided a measurement object measurement carrier, a measurement method, and a measurement kit that are useful for disease diagnosis and the like.

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Abstract

L'invention concerne un support ou un support lié par molécules de capture, destiné à s'utiliser dans la détermination d'un analyte dans un échantillon; un procédé de détermination utilisant le support ou le support lié par molécules de capture; un réactif destiné à la détermination d'un analyte, y compris le support ou le support lié par molécules de capture; et un processus de fabrication du support ou du support lié par molécules de capture. Un support comprend un substrat qui comporte à sa surface une région intégrée composée de deux ou plusieurs polymères qui possèdent des stéréo-régularités différentes et une substance de liaison de molécules fixée à la région intégrée du substrat; un support lié par molécules de capture qui comprend le support et une molécule de capture liée au support via la substance de liaison de molécules; un réactif comprenant le support ou le support lié par molécules de capture; et un procédé pour déterminer un analyte au moyen du support ou du support lié par molécules de capture.
PCT/JP2006/302993 2005-02-21 2006-02-21 Support destine a l'utilisation analytique et procede de determination utilisant ce support Ceased WO2006088192A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010522069A (ja) * 2007-03-19 2010-07-01 センター フォー アプライド プロテオミックス アンド モレキュラー メディシン バイオマーカーの収集のためのスマートヒドロゲル粒子
WO2020225971A1 (fr) * 2019-05-08 2020-11-12 株式会社日立ハイテク Procédé de prétraitement de dispositif d'analyse automatique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004180569A (ja) * 2002-12-03 2004-07-02 Toray Ind Inc 細胞分離カラム
JP2004271514A (ja) * 2003-02-18 2004-09-30 Fuji Photo Film Co Ltd バイオセンサー
JP2005189222A (ja) * 2003-12-04 2005-07-14 Fuji Photo Film Co Ltd センサー用固体基板
JP2005283396A (ja) * 2004-03-30 2005-10-13 Fuji Photo Film Co Ltd バイオセンサー

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004180569A (ja) * 2002-12-03 2004-07-02 Toray Ind Inc 細胞分離カラム
JP2004271514A (ja) * 2003-02-18 2004-09-30 Fuji Photo Film Co Ltd バイオセンサー
JP2005189222A (ja) * 2003-12-04 2005-07-14 Fuji Photo Film Co Ltd センサー用固体基板
JP2005283396A (ja) * 2004-03-30 2005-10-13 Fuji Photo Film Co Ltd バイオセンサー

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010522069A (ja) * 2007-03-19 2010-07-01 センター フォー アプライド プロテオミックス アンド モレキュラー メディシン バイオマーカーの収集のためのスマートヒドロゲル粒子
WO2020225971A1 (fr) * 2019-05-08 2020-11-12 株式会社日立ハイテク Procédé de prétraitement de dispositif d'analyse automatique
JPWO2020225971A1 (fr) * 2019-05-08 2020-11-12
JP7520824B2 (ja) 2019-05-08 2024-07-23 株式会社日立ハイテク 自動分析装置の前処理方法
US12422446B2 (en) 2019-05-08 2025-09-23 Hitachi High-Tech Corporation Pretreatment method of an automatic analyzer

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