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WO2013001895A1 - Support pour une immobilisation de matière en vue d'une utilisation en immuno-essai - Google Patents

Support pour une immobilisation de matière en vue d'une utilisation en immuno-essai Download PDF

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Publication number
WO2013001895A1
WO2013001895A1 PCT/JP2012/060223 JP2012060223W WO2013001895A1 WO 2013001895 A1 WO2013001895 A1 WO 2013001895A1 JP 2012060223 W JP2012060223 W JP 2012060223W WO 2013001895 A1 WO2013001895 A1 WO 2013001895A1
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Prior art keywords
substance
immobilized
carrier
antibody
group
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English (en)
Japanese (ja)
Inventor
則彦 大河内
弘人 菖蒲
裕一 田中
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Dai Nippon Printing Co Ltd
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Dai Nippon Printing Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

  • the present invention relates to a solid phase carrier for use in an immunoassay.
  • Enzyme-Linked Immunosorbent Assay (hereinafter referred to as ELISA), which is a kind of immunoassay, is used in a wide range of fields including drug discovery, diagnosis, environmental measurement, and food.
  • a typical ELISA includes (1) antibody or antigen immobilization (solid phase), (2) binding of a target substance, (3) binding of an enzyme-labeled antibody, (4) enzymatic reaction, (5) optical detection. It consists of five steps (absorption, fluorescence, luminescence). Horseradish peroxidase or alkaline phosphatase is used as the labeling enzyme.
  • ELISA is particularly sensitive among many immunoassays.
  • Patent Document 1 discloses a method of immobilizing an antibody on a gold surface via a PEG linker. Specifically, a nonionic functional group is introduced on the gold surface, one end of the heterobifunctional PEG is covalently bonded thereto, and the antibody is bonded to another end. It is described that noise in the surface plasmon resonance method is significantly reduced by this method.
  • Patent Document 2 discloses a method of immobilizing nucleic acids and proteins on a glass surface via a PEG linker. Specifically, a silane compound having a PEG linker is synthesized, applied to a glass surface, and then a nucleic acid or protein is bonded to the end of the PEG linker. It is described that this method improves the S / N ratio (sensitivity) of a biochip.
  • Patent Document 3 discloses a method of immobilizing a nucleic acid on a glass surface via a PEG linker. Specifically, an amino group is introduced by applying a silane coupling agent to the glass surface, one end of the homobifunctional PEG is covalently bonded, and a nucleic acid is bonded to another end. . It is described that this method improves the sensitivity of the biosensor.
  • the immobilization substance that is a biological substance is immobilized on the substance immobilization carrier having a PEG linker. It was difficult to immobilize the substance at a high density while maintaining its activity.
  • the present invention has been made in view of such circumstances, and a substance-immobilizing carrier having a PEG linker that realizes high detection sensitivity in an immunoassay such as ELISA, and immobilization of the substance to be immobilized on the carrier It is an object of the present invention to provide a method, and an immunoassay method using the carrier and a kit therefor.
  • the present inventor has found that a high detection sensitivity can be realized when an immunoassay is performed using a support having a hydrophilic polymer layer containing a PEG linker that satisfies specific conditions on the surface as a substance immobilization carrier, The present invention has been completed.
  • the present invention includes the following group of inventions.
  • a substance immobilization carrier for use in an immunoassay A support; At least a hydrophilic polymer layer disposed on the surface of the support, The hydrophilic polymer layer is A polyethylene glycol chain having a number average molecular weight of 176 or more; A substance immobilization carrier comprising an immobilized substance that is an antigen or an antibody that binds to a target substance, or a functional group linked to the polyethylene glycol chain that can form a covalent bond with the immobilized substance that is a target substance. .
  • the functional group includes a functional group containing n nitrogen atoms,
  • the nitrogen concentration in the hydrophilic polymer layer is 0.010 or more and 0.050 ⁇ n or less when the carbon concentration derived from the C—O bond in the hydrophilic polymer layer is 1.
  • the substance immobilizing carrier is 0.010 or more and 0.050 ⁇ n or less when the carbon concentration derived from the C—O bond in the hydrophilic polymer layer is 1.
  • a substance-immobilized carrier for use in an immunoassay A support; A hydrophilic polymer layer disposed on the surface of the support; At least an immobilized substance that is an antigen or an antibody that binds to a target substance or an immobilized substance that is a target substance,
  • the hydrophilic polymer layer includes a polyethylene glycol chain having a number average molecular weight of 176 or more, A substance-immobilized carrier in which the polyethylene glycol chain and the substance to be immobilized are linked via a covalent bond.
  • a method for producing a substance-immobilized carrier according to (4) or (5) A substance immobilization substance contacting step in which the substance immobilization support according to any one of (1) to (3) is brought into contact with a solution in which the substance to be immobilized is dissolved; A dry concentration step of drying and concentrating the solution in contact with the substance immobilization carrier; and Including methods.
  • a method for measuring a target substance by an immunoassay comprising the step of measuring the target substance using the substance-immobilized carrier of (4) or (5).
  • a kit for measuring a target substance by an immunoassay comprising the substance immobilization carrier according to any one of (1) to (3).
  • a kit for measuring a target substance by immunoassay comprising the substance-immobilized carrier according to (4) or (5).
  • the kit according to (13) for use in a sandwich-type immunoassay The substance-immobilized carrier according to (4) or (5), wherein the substance to be immobilized is an antigen or an antibody that binds to a target substance; A detectable antigen or detectable antibody that is a directly or indirectly detectable antigen or antibody that binds to the target substance non-competitively with the immobilized substance; A kit comprising at least
  • kits according to (13) for use in a direct competitive immunoassay The substance-immobilized carrier according to (4) or (5), wherein the substance to be immobilized is an antigen or an antibody that binds to a target substance;
  • a kit comprising at least a detectable target substance, which is a target substance detectable directly or indirectly, which binds to the immobilized substance competitively with a target substance in a sample to be measured.
  • a substance immobilization carrier for use in an immunoassay A support; At least a hydrophilic polymer layer disposed on the surface of the support, The hydrophilic polymer layer is A polyethylene glycol chain; A functional group linked to the polyethylene glycol chain capable of forming a covalent bond with an immobilized substance that is an antigen or an antibody that binds to a target substance or an immobilized substance that is a target substance,
  • the functional group comprises a functional group containing n nitrogen atoms;
  • the nitrogen concentration in the hydrophilic polymer layer is 0.010 or more and 0.050 ⁇ n or less when the carbon concentration derived from the C—O bond in the hydrophilic polymer layer is 1.
  • Substance immobilization carrier is 0.010 or more and 0.050 ⁇ n or less when the carbon concentration derived from the C—O bond in the hydrophilic polymer layer is 1.
  • a kit for measuring a target substance by an immunoassay comprising the substance immobilization carrier according to (17).
  • High detection sensitivity can be realized by performing an immunoassay using the substance immobilization carrier of the present invention.
  • FIG. 1 shows one embodiment of the present invention relating to a substance immobilizing carrier.
  • FIG. 2 shows an embodiment of the present invention relating to a method for producing a substance immobilizing carrier.
  • FIG. 3 shows an embodiment of an ELISA (indirect method) using the substance-immobilized carrier of the present invention.
  • FIG. 4 shows a calibration curve of ELISA (indirect method) using the substance-immobilized carrier of the present invention.
  • FIG. 5 shows an embodiment of an ELISA (sandwich method) using the substance-immobilized carrier of the present invention.
  • FIG. 6 shows a calibration curve of ELISA (sandwich method) using the substance-immobilized carrier of the present invention.
  • FIG. 1 shows one embodiment of the present invention relating to a substance immobilizing carrier.
  • FIG. 2 shows an embodiment of the present invention relating to a method for producing a substance immobilizing carrier.
  • FIG. 3 shows an embodiment of an ELISA (indirect method) using the substance-immobil
  • FIG. 7-1 shows a calibration curve of ELISA (sandwich method) when the trehalose concentration of the immobilized substance solution in Example 7 is 0% (w / v).
  • 7-2 shows the calibration curve of ELISA (sandwich method) when the trehalose concentration of the immobilized substance solution in Example 7 is 0.6% (w / v).
  • FIG. 7-3 shows the calibration curve of ELISA (sandwich method) when the trehalose concentration of the immobilized substance solution in Example 7 is 1.25% (w / v).
  • FIG. 7-4 shows a calibration curve of ELISA (sandwich method) when the trehalose concentration of the immobilized substance solution in Example 7 is 2.5% (w / v).
  • FIG. 7-5 shows an ELISA (sandwich method) calibration curve when the trehalose concentration of the immobilized substance solution in Example 7 is 5% (w / v).
  • FIG. 7-6 shows a calibration curve of ELISA (sandwich method) when the trehalose concentration of the immobilized substance solution in Example 7 is 10% (w / v).
  • FIG. 8 schematically shows an example of a reaction system for sandwich ELISA.
  • FIG. 9 schematically shows an example of a reaction system of a direct competitive ELISA.
  • FIG. 10 schematically shows an example of a reaction system for indirect competitive ELISA.
  • FIG. 11 shows an outline of an example of a method for producing the substance immobilizing carrier of the present invention.
  • FIG. 12 shows an outline of an example of the method for producing the substance-immobilized carrier of the present invention.
  • FIG. 13 schematically shows an example of an embodiment of the substance immobilizing carrier of the present invention.
  • the material and shape of the support in the present invention are not particularly limited as long as the material and shape can be used as a solid phase support in an immunoassay.
  • the material for the support include plastic, glass, quartz, silicon, and metal.
  • Plastic is particularly preferred as a support material for an immunoassay carrier because it is easy to mold and has few problems in transportation and disposal. That is, the support preferably used in the present invention contains plastic in all or at least part thereof.
  • the surface of the support on the side on which the hydrophilic polymer layer described later is formed preferably contains plastic, and more preferably the surface is made of plastic.
  • the plastic include polystyrene, polypropylene, polyvinyl chloride, polyethylene, cyclic polyolefin, acrylic resin, and polyethylene terephthalate.
  • the support surface may be subjected to a hydrophilic treatment such as plasma treatment or corona treatment in advance.
  • the support may be any support that has a surface that can be used as a solid phase in an immunoassay, and the overall shape is not particularly limited.
  • a support in the form of a microwell plate a plate-like body having a plurality of recesses
  • particles, slides, tubes, capillaries, microchannels, or the like can be used.
  • supports in the form of microwell plates, in particular polystyrene microwell plates are useful.
  • a hydrophilic polymer layer is disposed on the surface of the support.
  • the hydrophilic polymer layer includes at least a polyethylene glycol chain (PEG chain).
  • PEG chain polyethylene glycol chain
  • “Polyethylene glycol chain (or PEG chain)” is represented by the following formula: — (CH 2 —CH 2 —O) m — (M is an integer indicating the degree of polymerization) Refers to the structure represented by Surprisingly, the number average molecular weight of the PEG chains in the hydrophilic polymer layer affects the sensitivity of the immunoassay.
  • the number average molecular weight of the PEG chain is preferably 176 or more (m is 4 or more), more preferably 362 or more.
  • m is 4 or more
  • 362 the number average molecular weight of the PEG chain is smaller than the above lower limit, sufficient sensitivity may not be achieved as confirmed in experimental examples.
  • the upper limit of the number average molecular weight of the PEG chain is not particularly limited, but the number average molecular weight of the PEG chain is difficult to handle because the viscosity increases as the number average molecular weight increases and the arrangement of the PEG chains at high density is difficult. Is preferably 25000 or less, more preferably 10,000 or less.
  • the number average molecular weight of the PEG chain is PEG used as a raw material or PEG dissociated from a carrier: HO— (CH 2 —CH 2 —O) m —H (M is an integer indicating the degree of polymerization)
  • M is an integer indicating the degree of polymerization
  • the molecular weight of H 2 O (18.015) can be subtracted from the number average molecular weight of
  • the PEG number average molecular weight is determined by the vapor pressure osmotic pressure method or the membrane osmotic pressure method.
  • the vapor pressure osmotic pressure method can be used when the number average molecular weight of PEG is less than 100,000.
  • the membrane osmotic pressure method can be used when the number average molecular weight of PEG is 10,000 to 1,000,000.
  • At least one functional group capable of forming a covalent bond with the substance to be immobilized is directly or indirectly linked to one end of the PEG chain in the substance immobilization carrier before immobilization.
  • a functional group include (1H-imidazol-1-yl) carbonyl group, succinimidyloxycarbonyl group, epoxy group, aldehyde group, amino group capable of forming a covalent bond with the substance to be immobilized.
  • (1H-imidazol-1-yl) carbonyl group and succinimidyloxycarbonyl group are preferred.
  • These functional groups can react with a functional group such as an amino group of the substance to be immobilized to form a covalent bond.
  • the binding density of the PEG chain also affects the detection sensitivity of the immunoassay.
  • the bond density of PEG chains can be estimated to some extent using X-ray photoelectron spectroscopy (XPS).
  • XPS X-ray photoelectron spectroscopy
  • the ethylene glycol unit (CH 2 —CH 2 —O) gives the C—O component of the C (1s) signal in (XPS), such as (1H-imidazol-1-yl) carbonyl group, succinimidyloxycarbonyl group, etc.
  • This nitrogen atom-containing functional group gives an N (1s) signal in XPS.
  • the element concentration ratio N (1s) / CO has a clear correlation with the bond density of PEG chains.
  • the nitrogen concentration (N (1s) / C—O) obtained by XPS, where the carbon concentration derived from the C—O bond in the hydrophilic polymer layer is 1, is the same as described above. A numerical range is preferred.
  • the hydrophilic polymer layer may further contain a PEG chain to which no functional group is added or another hydrophilic compound.
  • a hydrophilic polymer layer is formed on the surface of the support at a position used as a solid phase in an immunoassay.
  • the hydrophilic polymer layer can be immobilized on the support through a covalent bond with a functional group chemically or physically immobilized on the support surface.
  • the carrier for immobilizing a substance of the present invention is a step S1101 for introducing a functional group onto the surface of a support using a primer layer or a coupling agent, and a PEG chain is linked to the functional group. It can be produced by a method including step S1102 and step S1103 of linking a functional group to the PEG chain end.
  • a primer layer is preferably formed on the surface, and a hydrophilic polymer layer is preferably formed on the surface of the primer layer.
  • the functional group on the side chain of the polysiloxane of the primer layer forms a covalent bond with the PEG chain end.
  • the substance immobilization carrier 10 includes a support 11 containing a plastic on the surface S, a primer layer 12 containing polysiloxane disposed on the surface S, and a hydrophilic material containing PEG chains disposed on the primer layer 12.
  • a functional polymer layer 13 is
  • the PEG chain — (CH 2 —CH 2 —O) m — in the hydrophilic polymer layer 13 is linked to the polysiloxane side chain A constituting the primer layer 12 through a covalent bond.
  • the side chain A is a group derived from R 1 of a silanol compound of formula 1 described later, and the functional group on R 1 or a functional group derived from the functional group is a hydroxyl group at the end of the PEG chain. Refers to a divalent group formed by forming a covalent bond with.
  • the polysiloxane in the primer layer 12 may be linear, or may have a branched or network structure, but preferably has a branched or network structure.
  • X is a bridging group bonded to a silicon atom of another repeating unit (not shown).
  • X as the bridging group includes an ether group (—O—) derived from the hydroxyl group of the silanol compound of Formula 1.
  • X is a monovalent group such as R 1 or R 2 defined in Formula 1, an unreacted hydroxyl group, a group Y defined in Formula 2 remaining without hydrolysis. It is the basis of.
  • a functional group R 3 capable of forming a covalent bond with the substance to be immobilized is directly or, if necessary, via a linker at the end of the PEG chain that is not linked to polysiloxane. Are preferably indirectly connected.
  • Q represents a bond or a linker.
  • the support 11 includes plastic on at least the surface S.
  • the primer layer 12 containing polysiloxane can be bonded to the surface S of the support 11 by physical adsorption. It is believed that physisorption is caused by van der Waals forces or hydrophobic interactions. Since it is not necessary to form a chemical bond such as a covalent bond between the primer layer 12 and the surface S of the support 11, the surface S is made of a plastic that does not contain a reactive functional group such as polystyrene. Even the primer layer 12 can be bonded.
  • the state of the polysiloxane in the primer layer 12 is not necessarily clear, as shown in FIG. 2, the main chain portions of a plurality of polysiloxane molecules are associated with each other, and the organic groups as side chains are formed on the support surface and the hydrophilic layer. There is a possibility that a two-layer structure facing the side is formed. The mechanism by which such a double structure is formed is presumed as follows. First, polysiloxane having increased van der Waals force due to the polymerization of silanol is physically adsorbed on the plastic surface. At this time, the organic group of the silanol compound is oriented to the plastic side by a hydrophobic interaction acting between the plastic surface and the organic group of the silanol compound.
  • the polysiloxane After the first layer of polysiloxane is formed, another polysiloxane is bonded to silanol groups (Si—OH) oriented on the solvent side. At this time, the organic group of the silanol compound is oriented to the solvent side by hydrogen bonding between the silanol groups. As a result, the polysiloxane is considered to have a two-layer structure as shown in FIG. The organic group oriented on the solvent side can form a covalent bond with the hydrophilic layer.
  • silanol groups Si—OH
  • the primer layer can be formed of a layer containing at least polysiloxane.
  • the polysiloxane is a polymer composed of repeating units of siloxane bonds (Si—O—Si) and can be obtained by condensation polymerization of a silanol compound.
  • the condensation of the silanol compound is a reaction that occurs between the molecules of the silanol compound.
  • the plastic molecule on the support surface does not have a reactive functional group, no reaction occurs between the silanol compound and the plastic molecule on the support surface. That is, the silanol compound and the formed polysiloxane are not physically reacted with the plastic molecules on the support surface, but are merely physically adsorbed.
  • silanol compound The silanol compound used in the present invention has an organic group containing a carbon atom directly connected to a silicon atom and having a functional group in addition to a silanol group (Si—OH). This organic group becomes the side chain of the polysiloxane.
  • Silanol compounds typically have a structure represented by Formula 1: (R 1 ) p (R 2 ) 4-pq Si (OH) q ... (Formula 1) (P is 1 or 2, q is 2 or 3, p + q is 3 or 4, and R 1 is an organic group containing a carbon atom directly connected to a silicon atom and having a functional group.
  • R 1 is preferably substituted with a functional group having one or more (preferably one) hydrogen atom through an appropriate linker structure as necessary, and has 1 to 20 carbon atoms, preferably 1 to 15, more preferably 1 to 10, particularly preferably 1 to 6 hydrocarbon groups (provided that all or part of the hydrocarbon groups are vinyl groups, such as the hydrocarbon group itself) If is a functional group, it need not be substituted by a functional group).
  • the hydrocarbon group is a saturated or unsaturated aliphatic hydrocarbon group (an alkyl group, an alkenyl group having 2 or more carbon atoms, or an alkynyl group having 2 or more carbon atoms) having a linear or branched chain or ring structure.
  • It may be a monocyclic or polycyclic aromatic hydrocarbon group having 6 or more carbon atoms, or the aromatic hydrocarbon group substituted by one or more aliphatic hydrocarbon groups.
  • the aliphatic hydrocarbon group may be substituted with one or more aromatic hydrocarbon groups.
  • the carbon-carbon bond may be interrupted by 1 or 2 identical or different atoms selected from oxygen, nitrogen and sulfur.
  • Preferred examples of the hydrocarbon group include a propyl group and an ethyl group.
  • the functional group for substituting one or more hydrogens of the hydrocarbon group in R 1 through an appropriate linker structure as necessary, it can react with a hydroxyl group of PEG to form a covalent bond.
  • the functional group is not particularly limited as long as it is a functional group that can be converted into a functional group that can react with a hydroxyl group of PEG to form a covalent bond, but typically (1H-imidazol-1-yl ) Carbonyl group, succinimidyloxycarbonyl group, glycidyl group, epoxy group, aldehyde group, amino group, thiol group, carboxyl group, azide group, cyano group, active ester group (1H-benzotriazol-1-yloxycarbonyl) Group, pentafluorophenyloxycarbonyl group, paranitrophenyloxycarbonyl group, etc.), halogenated carbonyl group Isocyanate group, a maleimide group, and the like.
  • a glycidyl group or an epoxy group is preferable.
  • the glycidyl group or epoxy group itself can react with the hydroxyl group of PEG to form a covalent bond, but the glycidyl group or epoxy group is converted into an aldehyde group according to the method described in JP-A-2009-156864.
  • the aldehyde group thus formed may be reacted with the hydroxyl group of PEG.
  • These functional groups may be substituted directly on the hydrogen atom of the hydrocarbon group or may be substituted via an appropriate linker structure. Examples of the linker structure include a divalent group having 0 to 3 carbon atoms and 0 to 3 identical or different heteroatoms selected from nitrogen, oxygen and sulfur.
  • R 1 include a 3-glycidoxypropyl group and a 2- (3,4-epoxycyclohexyl) ethyl group.
  • R 2 is preferably a hydrocarbon group similar to that described above for R 1 except that it is not substituted by a substituent (but selected independently of R 1 ), A linear or branched alkyl group having 1 to 6 carbon atoms is preferable, and a methyl group or an ethyl group is particularly preferable.
  • the silanol compound can be produced by hydrolyzing a silicon compound having a group capable of producing a silanol group (Si—OH) by hydrolysis.
  • silicon compounds have a structure represented by Formula 2: (R 1 ) p (R 2 ) 4-pq Si (Y) q ... (Formula 2) (Y is a group that can independently generate a silanol group by hydrolysis, and p, q, R 1 , and R 2 are as defined for the silanol compound).
  • Y is preferably an alkoxy group, a halogen atom, an aryloxy group, an alkoxy group substituted by an alkoxy group or an aryloxy group, an aryloxy group substituted by an alkoxy group or an aryloxy group, an alkylcarbonyloxy group, or the like.
  • Y particularly represents an alkoxy group having 1 to 6 carbon atoms (particularly a methoxy group, ethoxy group, isopropoxy group, tert-butoxy group), an alkoxy group having 1 to 6 carbon atoms substituted by an alkoxy group (for example, methoxyethoxy group). Group), an alkylcarbonyloxy group having 1 to 6 carbon atoms (for example, an acetoxy group), and a chlorine atom are preferable.
  • silicon compound of the formula 2 a commercially available compound as a silane coupling agent can be suitably used, and 3-glycidoxypropyltrimethoxysilane or 3-glycidoxypropyltriethoxysilane is particularly preferable.
  • the primer layer containing polysiloxane can be formed by a method including a step (primer layer forming step) of polymerizing the silanol compound of Formula 1 on the surface of the support.
  • the step preferably comprises hydrolyzing the silicon compound of formula 2 as follows to produce a silanol compound of formula 1 and a solution in which the produced silanol compound and base are dissolved in alcohol. Contacting on the surface of the body. Hydrolysis conditions are not particularly limited, but for example, the following method is possible.
  • dilute hydrochloric acid is added to the silicon compound of formula 2 to hydrolyze the group Y.
  • the pH of dilute hydrochloric acid is desirably adjusted to 2.0 to 3.0.
  • the molar ratio of water molecules to silicon compounds is 2-4.
  • the group Y is converted to a silanol group, and a silanol compound of the formula 1 is formed.
  • a silanol compound is applied to the support surface, and polysiloxane is formed by condensation polymerization.
  • the silanol compound of Formula 1 is soluble in alcohol along with the base.
  • the final concentration of the silanol compound is desirably adjusted within the range of 0.1 to 10% (v / v).
  • the base triethylamine, N, N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine and the like can be used, but not limited thereto. It is desirable to adjust the final concentration of the base in the range of 0.1 to 10% (v / v).
  • This silanol compound solution is brought into contact with the plastic surface of the support and allowed to stand for 10 minutes to 24 hours.
  • the reaction temperature can be set in the range of 4 to 80 ° C, but room temperature (20 to 25 ° C) is particularly preferable.
  • a primer layer containing polysiloxane is formed on the plastic surface by physical adsorption.
  • the coating density of the primer layer can be controlled by the concentration of silanol or base, or the time for which the silanol solution is brought into contact with the plastic surface. The higher the coating density of the primer layer, the higher the binding density of the PEG chain that is covalently bonded in the next step.
  • the functional group from the silanol compound on the side chain of the formed polysiloxane is derivatized and converted to another functional group
  • the functional group from the silanol compound on the side chain of the polysiloxane is subsequently formed after the primer layer is formed.
  • a derivatization step is performed in which is converted to a functional group capable of reacting with the hydroxyl group of PEG to form a covalent bond.
  • the support is a support containing glass, quartz or silicon on the surface, an organic group containing a carbon atom directly bonded to a silicon atom and having a functional group formed on the surface by hydrolysis of a silane coupling agent It is shared with PEG on the surface of the support by linking a silanol compound having a functional group or, if necessary, by derivatization that converts the functional group into another functional group capable of forming a covalent bond with PEG. Functional groups capable of forming bonds can be introduced.
  • the silane coupling agent and the PEG chain of the hydrophilic polymer layer can be linked by a covalent bond by a reaction between the functional group and one end of PEG.
  • silanol compound those similar to the silanol compound of the formula 1 described above with respect to the primer layer can be used.
  • silane coupling agent those similar to the silicon compound of formula 2 described above with respect to the primer layer can be used.
  • a functional group on an organic group derived from a silanol compound or a silane coupling agent or other functional group derived from the functional group introduced on the support surface is covalently bonded to a hydroxyl group at the PEG end.
  • PEG is reacted to form a PEG chain on the surface of the support into which a functional group has been introduced by a primer layer, a silane coupling agent or the like (S1102).
  • a PEG containing a catalytic amount of concentrated sulfuric acid is brought into contact.
  • PEG having a number average molecular weight exceeding 1000 is previously heated and melted.
  • PEG may be diluted with tert-butyl alcohol or the like.
  • the PEG solution is brought into contact with the plastic surface and heated.
  • the heating temperature can be set in the range of 60 to 100 ° C., but considering the heat resistance of the plastic, it is preferably around 80 ° C. (75 ° C.
  • the heating time can be set in the range of 10 minutes to 24 hours, but when the heating temperature is around 80 ° C., 10 to 60 minutes is preferable.
  • the PEG chain is covalently bonded to the primer layer.
  • the binding density of the PEG chain depends on the coating density of the primer layer.
  • At least one functional group capable of forming a covalent bond with the substance to be immobilized is directly or indirectly linked to one end of the PEG chain (S1103).
  • the method for introducing the functional group is not particularly limited.
  • a preferred embodiment of the method for introducing the (1H-imidazol-1-yl) carbonyl group and the succinimidyloxycarbonyl group as a substituent for substituting the hydrogen of the hydroxyl group at the end of the PEG chain is as follows.
  • CDI 1,1′-carbonyldiimidazole
  • DSC di (N-succinimidyl) carbonate
  • the above reaction needs to be carried out in an organic solvent containing almost no moisture. Since plastics generally have low resistance to organic solvents, when the support contains plastics, it is preferable to use acetonitrile, dimethyl sulfoxide, or a mixed solvent obtained by mixing these organic solvents in an appropriate ratio.
  • the water content of these organic solvents is desirably 0.1% by weight or less.
  • the final concentration of CDI or DSC can be set in the range of 0.01 to 1M. However, when the reaction is performed at room temperature or lower, it is preferably 0.1M or higher.
  • the support contains plastic it is desirable to set the reaction temperature in the range of 4 to 25 ° C. in order to avoid damage to the plastic.
  • the reaction time is preferably set in the range of 10 minutes to 24 hours, and preferably 10 minutes to 60 minutes when the CDI concentration is around 0.5 M (0.4 to 0.6 M).
  • an antigen or antibody that binds to a target substance or a target substance is immobilized on the substance immobilization carrier of the present invention, depending on the target immunoassay.
  • these target substances for immobilization are called “substances to be immobilized”.
  • an antigen or antibody that binds to a target substance is an immobilized substance (sandwich method or direct competition method described later)
  • the combination of the immobilized substance and the target substance is specific based on an antigen-antibody reaction.
  • the combination is not particularly limited as long as it is a combination that can be easily combined.
  • the immobilized substance when the target substance is an antigen (including a hapten), can be an antibody (including an antibody fragment), and when the target substance is an antibody (including an antibody fragment)
  • the immobilized substance can be an antigen (including a hapten).
  • the target substance is an immobilized substance (indirect competition method described later)
  • the target substance is an antigen (including a hapten) or an antibody (including an antibody fragment).
  • the antigen as the immobilized substance and / or the target substance is not particularly limited as long as it is a substance exhibiting specific antigen-antibody reactivity with an antibody.
  • Typical antigens include natural antigens such as proteins, peptides, saccharides, nucleic acids (DNA, RNA), lipids, coenzymes, cells, viruses, bacteria, and complexes of these, derivatives of natural antigens, and artificial synthesis. Hapten, artificial antigen and the like.
  • An antibody as an immobilized substance and / or a target substance refers to an immunoglobulin exhibiting specific antigen-antibody reactivity to a certain antigen and a fragment thereof, and may be subjected to chemical modification or the like as necessary. .
  • the substance to be immobilized is not particularly limited as long as it has a functional group capable of reacting with a functional group of the PEG chain to form a covalent bond.
  • Such functional groups typically include amino groups, but also include thiol groups, carboxyl groups, hydroxyl groups, alkoxides, secondary amines, tertiary amines, azide groups, cyano groups, and the like. Even if the substance to be immobilized has no functional group capable of forming a covalent bond by reaction with a functional group linked to the PEG chain, such as an amino group, the amino group is not present in these substances. Etc. can be used for immobilization by artificially introducing the above.
  • the immobilization of the substance to be immobilized is, for example, as shown schematically in FIG. 12, a step S1201 of bringing the substance immobilization carrier of the present invention into contact with a solution in which the substance to be immobilized is dissolved, and drying and concentrating the solution.
  • step S1203 for inactivating the functional group at the end of the PEG chain.
  • the substance to be immobilized is dissolved in a buffer solution.
  • a buffer solution that does not contain an amino group component.
  • a phosphate buffer solution or a carbonate-bicarbonate buffer solution can be used.
  • the final concentration of the substance to be immobilized is preferably adjusted in the range of 0.01 to 10 mg / ml.
  • This solution to be immobilized is brought into contact with a carrier (S1201), and then dried and concentrated as it is (S1202). Dry concentration increases the concentration of the substance to be immobilized in the reaction system, so that immobilization of the substance to be immobilized is significantly promoted.
  • the concentration of the substance to be immobilized in the reaction system can be increased, and the immobilization reaction can be promoted.
  • the substance to be immobilized is a protein such as an antigen or an antibody
  • the protein may be denatured by drying.
  • sugars such as trehalose, sucrose, lactose, and maltose to the immobilized substance solution.
  • trehalose has an appropriate moisture retention, is easy to dry concentrate the solution, and because it is difficult to form coarse crystals during dry concentration, it can be uniformly dried and concentrated. preferable.
  • the final concentration of saccharide may be adjusted in the range of 1 to 20% (w / v), more preferably 1 to 10% (w / v).
  • Triton registered trademark
  • X-100 and Tween registered trademark It is preferred to add a nonionic surfactant such as 20.
  • the substance to be immobilized is thinly spread on the surface of the carrier, so that not only the amount of the substance to be immobilized can be saved, but also the time required for drying and concentration can be shortened.
  • the final concentration of the nonionic surfactant in the solution to be immobilized is preferably adjusted in the range of 0.01 to 0.1% (v / v).
  • a functional group such as an amino group contained in the substance to be immobilized reacts with a functional group linked to the PEG chain, and a covalent bond such as an amide bond or a urethane bond is formed.
  • the reaction temperature is preferably set in the range of 4 to 37 ° C. and the contact time in the range of 5 minutes to 24 hours. As a result, the substance to be immobilized is immobilized on the carrier via the PEG chain.
  • the unreacted functional group linked to the PEG chain is bound to a low molecular compound having an amino group, thereby making the functional group more reactive.
  • the surface of the carrier after reacting the functional group with the low molecular weight compound is desirably hydrophilic. This is because a hydrophilic surface generally has an effect of suppressing nonspecific adsorption of a biological substance.
  • a low molecular compound having an amino group and further having a hydrophilic group in addition to the amino group.
  • examples of such a low molecular weight compound include 2-aminoethanol and 2- (2-aminoethoxy) ethanol, and 2- (2-aminoethoxy) ethanol is particularly preferable.
  • This low molecular weight compound is dissolved in a buffer solution such as PBS so as to have a concentration of 10 to 1000 mM and brought into contact with a carrier on which a desired substance is already immobilized.
  • the reaction temperature is preferably set in the range of 4 to 37 ° C. and the reaction time in the range of 2 minutes to 24 hours.
  • the present invention also relates to an immunoassay comprising a step of measuring a target substance using the substance-immobilized carrier of the present invention.
  • “measuring a target substance” refers to measuring the presence or absence of the target substance in the measurement target sample and / or the amount of the target substance in the measurement target sample.
  • the immunoassay comprises the substance-immobilized carrier of the present invention and the sample to be measured on the substance to be immobilized on the carrier, and an amount of antigen correlated with the amount of the target substance in the sample to be measured.
  • an antibody wherein the antigen or antibody is a target substance in a sample to be measured, a detectable target substance added to the antigen-antibody reaction system as necessary, or a detection added to the antigen-antibody reaction system as needed
  • a step of detecting the antigen or antibody bound to the substance to be immobilized in the step
  • the immunoassay using the substance-immobilized carrier of the present invention can be performed by a known method, and can be in any mode such as a sandwich system, a direct competition system, an indirect competition system, and the like.
  • a substance-immobilized carrier in which an antigen or antibody that binds to a target substance is immobilized as a substance to be immobilized, and a target substance that binds to the target substance in a non-competitive manner are directly used.
  • At least a detectable antigen or detectable antibody that is a detectable or indirectly detectable antigen or antibody is used.
  • a substance-immobilized carrier 101 on which an antibody 102 that specifically binds to a target substance 103 that is an antigen is immobilized, and a labeled antibody 104 that is directly detectable and is labeled with a labeling substance (such as an enzyme).
  • a labeling substance such as an enzyme
  • Sandwich-type immunoassays are typically A primary reaction step (FIG. 8B), in which a measurement target sample is brought into contact with the substance-immobilized carrier, and an antigen-antibody reaction between a target substance and a target substance in the measurement target sample is performed. After the primary reaction step, the substance-immobilized carrier is brought into contact with the detectable antigen or antibody, and the antigen-antibody reaction between the target substance bound to the substance to be immobilized and the detectable antigen or detectable antibody A secondary reaction step (FIG. 8C), After the secondary reaction step, at least a detection step (FIG. 8D) for detecting the detectable antigen or the detectable antibody bound to the substance-immobilized carrier is included.
  • a detection step FIG. 8D
  • the primary reaction step and the secondary reaction step appropriately include a washing step for washing and removing components not immobilized on the carrier after the antigen-antibody reaction.
  • the “detectable antigen or detectable antibody” used in the secondary reaction step binds to the target substance at a position that does not compete with the immobilized substance.
  • the detectable antigen or detectable antibody may be a labeled antigen or labeled antibody (directly detectable antigen or antibody) linked to a detectable labeling substance, or a detectable labeling substance, and It may be an antigen or antibody (an indirectly detectable antigen or antibody) that can be linked and labeled by reaction. Examples of the latter include binding to an antigen or antibody capable of binding to a secondary antibody linked to a detectable labeling substance, or a labeling substance linked to one of the biotin-avidin (or biotin-streptavidin) binding pairs. Examples thereof include an antigen or an antibody linked to the other of the binding pair.
  • At least a detectable target substance, which is a directly or indirectly detectable target substance that binds to is used.
  • a substance-immobilized carrier 111 on which an antibody 112 that specifically binds to a target substance 113 that is an antigen is immobilized, and a labeling substance that binds competitively with the target substance 113 in the sample to be measured and the antibody 112 1 schematically shows an example of an immunoassay by a direct competitive method in which a target substance 114 (labeled antigen) that is directly detected and labeled with an enzyme or the like is used to measure the target substance 113 in a sample to be measured.
  • Immunoassays using a direct competition format typically A sample to be measured, a detectable target substance, and the substance-immobilized carrier are brought into contact with each other, an antigen-antibody reaction between the target substance in the sample to be measured and the immobilized substance, a detectable target substance and the target substance
  • a direct competitive reaction step (FIGS. 9B and 9C) for competitively performing an antigen-antibody reaction with an immobilized substance;
  • a detection step (FIG. 9 (e)) for detecting a detectable target substance bound to the substance-immobilized carrier is included.
  • the direct competitive reaction step appropriately includes a step (FIG. 9 (d)) of washing and removing the target substance not bound to the carrier after the antigen-antibody reaction and the detectable target substance.
  • the target substance is an antigen or antibody
  • the detectable target substance is a corresponding detectable antigen or detectable antibody.
  • the detectable target substance can be detected independently of the target substance in the sample to be measured, in the detectable antigen or detectable antibody described above with respect to the sandwich method, the target substance is the antigen or antibody. The used one can be used.
  • a substance-immobilized carrier in which a target substance is immobilized as an immobilized substance is usually directly coupled to a target substance in the measurement target sample and the immobilized substance.
  • at least a detectable antigen or antibody that is an indirectly detectable antigen or antibody is used.
  • the substance-immobilized carrier 121 on which the same immobilized substance 122 as the target substance 123 that is an antigen is immobilized, and the target substance 123 and the immobilized substance 122 in the sample to be measured are bound competitively.
  • An example of an immunoassay by an indirect competitive method in which a target substance 123 in a measurement target sample is measured using a primary antibody 124 that can be indirectly detected through a labeled secondary antibody 125 is schematically shown.
  • Immunoassays using an indirect competitive format typically A sample to be measured, a detectable antigen or a detectable antibody, and the substance-immobilized carrier are contacted, and an antigen-antibody reaction between the target substance in the sample to be measured and the detectable antigen or the detectable antibody, An indirect competitive reaction step (FIGS. 10 (b) and 10 (c)) for competitively performing an antigen-antibody reaction between the immobilized substance and the detectable antigen or detectable antibody; After the indirect competitive reaction step, at least a detection step (FIGS. 10 (e) and (f)) for detecting the detectable antigen or the detectable antibody bound to the substance-immobilized carrier is included.
  • the indirect competitive reaction step appropriately includes a step (FIG. 10 (d)) of washing and removing the target substance and the detectable antigen or detectable antibody that are not bound to the carrier after the antigen-antibody reaction.
  • detectable antigen or detectable antibody those similar to the detectable antigen or detectable antibody described above for the sandwich method can be used.
  • the means for detecting the antigen or antibody in the immunoassay using the substance-immobilized carrier of the present invention is not particularly limited, and an antigen or antibody labeled directly or indirectly with any labeling substance can be used.
  • an amplified detection signal such as an enzyme (ELISA method), a nucleic acid (immunoPCR), an electrochemiluminescent substance (electrochemiluminescent method), a fluorescent substance, a chemiluminescent substance, or a radioactive substance can be generated.
  • an amplified detection signal such as an enzyme (ELISA method), a nucleic acid (immunoPCR), an electrochemiluminescent substance (electrochemiluminescent method), a fluorescent substance, a chemiluminescent substance, or a radioactive substance can be generated.
  • Possible labeling substances are mentioned.
  • the immunoassay of the present invention is preferably an enzyme-linked immunosorbent assay (ELISA) that uses an enzyme as a labeling substance and performs detection based
  • the enzyme that can be used as the labeling substance is not particularly limited, and examples thereof include peroxidase such as horseradish peroxidase, ⁇ -galactosidase, alkaline phosphatase, and glucose oxidase.
  • detection methods using enzyme activity include detection methods using chemiluminescent substrates that chemiluminescent by enzyme activity, chromogenic substrates that generate color by enzyme activity, chemiluminescent substrates that emit chemical fluorescence by enzyme activity, etc.
  • a detection method using a luminescent substrate is preferred. When a chromogenic substrate is used, the enzyme reaction takes a considerable amount of time. This is because the concentration range of the target substance to be quantified in the present invention is as low as 1 to 2 digits.
  • chemiluminescent substrate Since detection using a chemiluminescent substrate is generally more sensitive than detection using a chromogenic substrate, the time required for the enzyme reaction can be shortened. In particular, if a highly sensitive chemiluminescent substrate used for detection of a trace amount of target substance at the femtogram level is used, a synergistic effect can be obtained in terms of sensitivity.
  • chemiluminescent substrates include ECL TM Advance (GE Healthcare), ECL TM Plus (GE Healthcare), Immunostar (registered trademark) LD (Wako Pure Chemical Industries), CDP-STAR (registered trademark) (Roche Diagnostics).
  • CSPD registered trademark
  • CSPD registered trademark
  • SuperSignal West Femto Maxum Sensitive Substrate enzymes for this purpose include peroxidases such as horseradish peroxidase and alkaline phosphatase. Is mentioned.
  • a detector for detecting chemiluminescence a luminescence plate reader or a CCD imager can be used. These detectors are also advantageous in that they have a wide dynamic range. That is, when the substance-immobilized carrier of the present invention and the chemiluminescent substrate are combined, an ELISA having a wide dynamic range can be achieved quickly, with high sensitivity.
  • the substance immobilization carrier of the present invention can constitute a kit for measuring a target substance by immunoassay.
  • the kit can further comprise components necessary for the immunoassay.
  • a directly or indirectly detectable antigen or antibody that binds to a target substance bound to an immobilized substance by an antigen-antibody reaction depending on the type of immunoassay, by the antigen-antibody reaction
  • examples include directly or indirectly detectable antigens or antibodies (including directly or indirectly detectable target substances) that bind to an immobilized substance by an antigen-antibody reaction.
  • components necessary for the immunoassay include a labeling substance capable of binding to the indirectly detectable antigen or antibody for use in indirect detection, and a detection reagent (for example, when the labeling substance is an enzyme).
  • a labeling substance capable of binding to the indirectly detectable antigen or antibody for use in indirect detection and a detection reagent (for example, when the labeling substance is an enzyme).
  • a chemiluminescent substrate that chemiluminescents by enzyme activity include a chemiluminescent substrate that chemiluminescents by enzyme activity, a chromogenic substrate that develops color by enzyme activity, a chemiluminescent substrate that emits chemical fluorescence by enzyme activity, and the like.
  • “Directly or indirectly detectable antigen or antibody” and “directly or indirectly detectable target substance” can be the same as those described in the above “Immunoassay” column.
  • the kit further includes a reagent used for immobilizing the substance to be immobilized on the substance immobilization carrier (for example, a buffer solution for dissolving the substance to be immobilized, in which sugars and nonionic surfactants are dissolved). ) May be included.
  • a reagent used for immobilizing the substance to be immobilized on the substance immobilization carrier for example, a buffer solution for dissolving the substance to be immobilized, in which sugars and nonionic surfactants are dissolved.
  • the substance-immobilized carrier of the present invention can constitute a kit for measuring a target substance by immunoassay.
  • the kit can further comprise components necessary for the immunoassay. Specific examples of components necessary for the immunoassay are as described above.
  • FIG. 1 shows an embodiment of the present invention relating to a substance immobilizing carrier.
  • a primer layer containing polysiloxane is formed on the surface of a support made of polystyrene.
  • One end of the PEG chain is covalently bonded to the primer layer.
  • the hydrophilic polymer layer has a condition that the number average molecular weight of the PEG chain is 176 to 25000, and a condition that the element concentration ratio N (1s) / CO in XPS is in the range of 0.010 to 0.100. At least one of the conditions is satisfied.
  • Example 2 A substance-immobilizing support was produced by the method shown in FIG. The specific procedure is described below.
  • Silanol was prepared by adding 0.35 ml of dilute hydrochloric acid (pH 2.4) to 1.65 ml of 3-glycidoxypropyltrimethoxysilane (Momentive Performance Materials). This was added to 100 ml of 2-propanol (Pure Chemical). To this, 4 ml of triethylamine (Wako Pure Chemical Industries) was further added. 100 ⁇ l of this silanol solution was dispensed into each well of a 96-well microplate (BD Falcon TM ). It was allowed to stand at room temperature for 75 minutes. Thereafter, the inside of the well was washed with pure water and dried by nitrogen blowing.
  • a primer layer containing polysiloxane and an epoxy group was formed in the well of the microplate.
  • 100 ⁇ l of PEG 4000 containing a catalytic amount of concentrated sulfuric acid (number average molecular weight 2700-3400, Kanto Chemical) was dispensed into each well.
  • the mixture was heated at 90 ° C. for 30 minutes. Thereafter, the inside of the well was washed with pure water and dried by nitrogen blowing.
  • a hydrophilic polymer layer containing PEG was formed on the primer layer.
  • a CDI (Tokyo Kasei) solution with a final concentration of 0.5 M is prepared using an equal weight mixed solvent of dehydrated acetonitrile (Kanto Chemical) and dehydrated dimethyl sulfoxide (Kanto Chemical), and 10 ⁇ l is dispensed into each well. did. It was allowed to stand at room temperature for 20 minutes. Thereafter, the inside of the well was washed with pure water and dried by nitrogen blowing. By this operation, a PEG derivative in which a (1H-imidazol-1-yl) carbonyl group was introduced at the end of PEG contained in the hydrophilic polymer layer was formed.
  • Example 3 Using the substance immobilization carrier (96-well microplate) of Example 2, ELISA (indirect method, antigen-antibody-antibody sandwich method) shown in FIG. 3 was performed. The specific procedure is described below.
  • Triton (registered trademark) Carbonate-bicarbonate buffer (pH 9.6) containing X-100 (Wako Pure Chemical Industries) was used as a solid phase buffer
  • 0.1% Triton (registered trademark) A phosphate buffer (PBS) containing X-100 and 0.5 M NaCl is used as a washing buffer
  • PBS containing 1% BSA is used as a dilution buffer.
  • lysozyme (Wako Pure Chemical Industries) was dissolved in a solid phase buffer to prepare a lysozyme solution having a final concentration of 50 ⁇ g / ml. 5 ⁇ l of this solution was dispensed into each well of the 96-well microplate of Example 2.
  • a carbonate-bicarbonate buffer (pH 9.6) is used as a solid phase buffer, 0.05% Tween (registered trademark).
  • PBS containing 20 is used as a washing buffer
  • PBS containing 1% BSA is used as a blocking buffer and a dilution buffer.
  • lysozyme (Wako Pure Chemical Industries) was dissolved in a solid phase buffer to prepare a lysozyme solution having a final concentration of 5 ⁇ g / ml. 100 ⁇ l of this solution was dispensed into each well of a 96-well microplate. After leaving at room temperature for 2 hours, the inside of the well was washed twice with a washing buffer.
  • Example 4 Using the 96-well microplate obtained in Example 2, the ELISA (antibody-antigen-antibody sandwich method) shown in FIG. 5 was performed. The specific procedure is described below.
  • a carbonate-bicarbonate buffer solution (pH 9.6) containing X-100 (Wako Pure Chemical Industries, Ltd.) is used as a solid phase buffer.
  • Anti-IL-1 ⁇ antibody (Biolegend) was dissolved in an immobilization buffer to prepare an antibody solution having a final concentration of 50 ⁇ g / ml. 5 ⁇ l of this solution was dispensed into each well of the 96-well microplate obtained in Example 2. After leaving at 37 ° C. for 2 hours to dry and concentrate, the well was washed twice with a washing buffer.
  • 0-2500 pg / ml IL-1 ⁇ (Wako Pure Chemical Industries) was prepared using a dilution buffer, and 50 ⁇ l was dispensed into each well. After leaving at room temperature for 60 minutes, the well was washed once with a washing buffer. A biotin-labeled secondary antibody (Biolegend) was diluted 500 times using a dilution buffer, and 50 ⁇ l was dispensed into each well. After leaving at room temperature for 30 minutes, the well was washed once with a washing buffer.
  • HRP-labeled streptavidin (Prozyme) was diluted 4000 times using a dilution buffer (final concentration: 0.25 ⁇ g / ml), and 50 ⁇ l was dispensed into each well. After leaving at room temperature for 10 minutes, the well was washed three times with a washing buffer. 30 ⁇ l of Immunostar LD (Wako Pure Chemical Industries), which is a chemiluminescent substrate, was added, and a chemiluminescent image was acquired using LAS4000mini (GE Healthcare). Finally, chemiluminescence intensity was calculated using dedicated software, and a calibration curve as shown in FIG. 6 (present invention) was created. The sensitivity was 0.15 pg / ml.
  • ELISA antibody-antigen-antibody
  • FIG. 5 ELISA (antibody-antigen-antibody) shown in FIG. 5 using an untreated 96-well microplate (BD Falcon TM ) and a conventional 96-well microplate (polystyrene 96-well microplate hydrophilized by oxygen plasma treatment) Sandwich method) was carried out. The specific procedure is described below.
  • pg / ml IL-1 ⁇ (Wako Pure Chemical Industries) was prepared using a dilution buffer, and 50 ⁇ l was dispensed into each well. After leaving at room temperature for 60 minutes, the well was washed once with a washing buffer. A biotin-labeled secondary antibody (Biolegend) was diluted 500 times using a dilution buffer, and 50 ⁇ l was dispensed into each well. After leaving at room temperature for 30 minutes, the well was washed once with a washing buffer.
  • HRP-labeled streptavidin (Prozyme) was diluted 4000 times using a dilution buffer (final concentration: 0.25 ⁇ g / ml), and 50 ⁇ l was dispensed into each well. After leaving at room temperature for 30 minutes, the well was washed three times with a washing buffer. 30 ⁇ l of Immunostar LD (Wako Pure Chemical Industries), which is a chemiluminescent substrate, was added, and a chemiluminescent image was acquired using LAS4000mini (GE Healthcare). Finally, chemiluminescence intensity was calculated using dedicated software, and a calibration curve as shown in FIG. 6 (unprocessed, conventional product) was created. The sensitivity of the untreated product and the conventional product was 9.8 pg / ml and 2.4 pg / ml, respectively.
  • Example 5 The influence of the silanol treatment time and the number average molecular weight of the PEG chain on the sensitivity of the ELISA (indirect method) was examined. The specific procedure is described below.
  • Silanol was prepared by adding 0.35 ml of dilute hydrochloric acid (pH 2.4) to 1.65 ml of 3-glycidoxypropyltrimethoxysilane (Momentive Performance Materials). This was added to 100 ml of 2-propanol (Pure Chemical). To this, 0.5 ml of triethylamine (Wako Pure Chemical Industries) was added. 100 ⁇ l of this silanol solution was dispensed into each well of a 96-well microplate (BD Falcon TM ). It was allowed to stand at room temperature for 60 to 135 minutes. Thereafter, the inside of the well was washed with pure water and dried by nitrogen blowing.
  • a primer layer containing polysiloxane and an epoxy group was formed in the well of the microplate.
  • 100 ⁇ l of PEG (13 types) containing a catalytic amount of concentrated sulfuric acid was dispensed into each well.
  • the mixture was heated at 80 ° C. for 45 minutes. Thereafter, the inside of the well was washed with pure water and dried by nitrogen blowing.
  • a hydrophilic polymer layer containing PEG was formed on the primer layer.
  • a CDI (Tokyo Kasei) solution with a final concentration of 0.5 M is prepared using an equal weight mixed solvent of dehydrated acetonitrile (Kanto Chemical) and dehydrated dimethyl sulfoxide (Kanto Chemical), and 20 ⁇ l is dispensed into each well. did. It was allowed to stand at room temperature for 30 minutes. Thereafter, the inside of the well was washed with pure water and dried by nitrogen blowing. By this operation, a PEG derivative in which a (1H-imidazol-1-yl) carbonyl group was introduced at the end of PEG contained in the hydrophilic polymer layer was formed.
  • the number average molecular weight of each raw material shown in Table 2 is as follows.
  • Example 6 The XPS analysis of the substance immobilization support obtained in Example 5 was performed.
  • the XPS analysis was performed using an X-ray spectroscopic analyzer “ESCA5600” manufactured by ULVAC-PHI and setting the photoelectron uptake angle to 45 °.
  • ESCA5600 X-ray spectroscopic analyzer manufactured by ULVAC-PHI
  • Table 2 a clear correlation was recognized between the sensitivity of ELISA and N (1s) / CO. That is, it has been found that when the value of N (1s) / CO is in the range of 0.010 to 0.100, a significantly higher sensitivity can be obtained than before.
  • Example 7 As described above, it is important to add trehalose to the buffer for the purpose of preventing protein denaturation due to drying.
  • Example 4 six types of buffers having different trehalose concentrations were prepared, and the trehalose concentration dependency in ELISA was examined. As a result, as shown in FIGS. 7-1 to 7-6, solidification time with 5 to 10% (w / v) trehalose (time from contact of the immobilized substance dissolving solution to completion of the drying concentration step) It was found that the decrease in signal associated with can be prevented.

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Abstract

La présente invention a pour but de procurer un support pour immobiliser une matière ayant un liant PEG qui exprime une sensibilité élevée de détection dans l'ELISA. La présente invention concerne un support pour immobiliser une matière en vue d'une utilisation dans un immuno-essai, et ledit support pour une immobilisation d'une matière comprend au moins un substrat et une couche de polymère hydrophile qui est positionnée sur la surface du substrat. La couche de polymère hydrophile contient des chaînes de polyéthylène glycol et des groupes fonctionnels qui sont liés aux chaînes de polyéthylène glycol, lesdits groupes fonctionnels étant aptes à former des liaisons covalentes avec la matière immobilisée, qui est un antigène ou un anticorps qui se lie avec une substance cible ou qui est une substance cible. La présente invention concerne également un procédé d'immobilisation d'une matière à immobiliser dans ledit support et sur un procédé et un coffret pour mettre en œuvre des mesures par des immuno-essais, dans lesquels ledit support est utilisé.
PCT/JP2012/060223 2011-06-28 2012-04-16 Support pour une immobilisation de matière en vue d'une utilisation en immuno-essai Ceased WO2013001895A1 (fr)

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