[go: up one dir, main page]

WO2017155019A1 - Inhibiteur d'adsorption de protéine et procédé d'inhibition d'adsorption de protéine - Google Patents

Inhibiteur d'adsorption de protéine et procédé d'inhibition d'adsorption de protéine Download PDF

Info

Publication number
WO2017155019A1
WO2017155019A1 PCT/JP2017/009405 JP2017009405W WO2017155019A1 WO 2017155019 A1 WO2017155019 A1 WO 2017155019A1 JP 2017009405 W JP2017009405 W JP 2017009405W WO 2017155019 A1 WO2017155019 A1 WO 2017155019A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein adsorption
water
protein
acrylic polymer
adsorption inhibitor
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/JP2017/009405
Other languages
English (en)
Japanese (ja)
Inventor
賢 田中
山田　智
伸行 坂元
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.)
Yamagata University NUC
NOF Corp
Original Assignee
Yamagata University NUC
NOF Corp
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 Yamagata University NUC, NOF Corp filed Critical Yamagata University NUC
Priority to JP2018504575A priority Critical patent/JP6803371B2/ja
Publication of WO2017155019A1 publication Critical patent/WO2017155019A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
    • C08F20/10—Esters
    • C08F20/26—Esters containing oxygen in addition to the carboxy oxygen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/04—Coating
    • C08J7/056—Forming hydrophilic coatings
    • 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
    • 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
    • G01N33/531—Production of immunochemical test materials

Definitions

  • the present invention relates to a non-specific adsorption inhibitor for proteins and a method for inhibiting adsorption.
  • a protein adsorption inhibitor that can prevent the adsorption of impurities (proteins) in a sample to the solid phase surface of a substrate such as an immune reaction container or a measurement instrument, and has a high washing resistance
  • a protein that can prevent the adsorption of impurities (proteins) in a sample to the solid phase surface of a substrate such as an immune reaction container or a measurement instrument, and has a high washing resistance
  • the present invention also relates to a method for suppressing adsorption, and further to a substrate treated with the adsorption inhibitor.
  • the detection method is being switched from a method using an enzyme reaction such as peroxidase or alkaline phosphatase to a method using fluorescence or chemiluminescence. Theoretically, it is said that the presence of one molecule to be inspected can be confirmed by using fluorescence or chemiluminescence as a detection method, but in reality, the desired sensitivity cannot be obtained.
  • the antibody to be measured, the antigen, or the label of these labels used for measurement such as the substrate of an immune reaction container or measuring instrument
  • Non-specific adsorption to the solid surface is mentioned.
  • a substance that coexists with multiple types of biomolecules such as serum, plasma, cell extract and urine
  • an unspecified number of coexisting substances representing various proteins are used as a base material for immune reaction containers and measuring instruments.
  • Generation of noise due to non-specific adsorption to the solid phase surface is also a factor preventing high sensitivity.
  • Non-Patent Document 1 and Patent Document 1 biologically-derived proteins such as bovine serum albumin, casein, and gelatin that have not been involved in an immune reaction are conventionally used as a buffer.
  • a method for suppressing non-specific adsorption of proteins involved in immune reactions by adsorbing biologically-derived proteins on a fixed surface of a substrate such as an immune reaction container or measuring instrument using a treatment solution as a solution. It is used.
  • Patent Document 2 contains 2-methacryloyloxyethyl phosphorylcholine polymer
  • Patent Document 3 contains glycosylethyl (meth) acrylate
  • Patent Document 4 contains an oxyalkylene group.
  • a chemical synthetic product as a protein adsorption inhibitor is physically adsorbed on a solid phase surface such as an immune reaction container or a measuring instrument, thereby producing an effect.
  • the adsorption suppression ability of the level of the non-specific adsorption inhibitor based on the above-described conventional technology may still be insufficient to suppress the generation of noise due to non-specific adsorption in the high sensitivity measurement.
  • biocompatible materials that are used in contact with bodily fluids such as blood or living tissue and can reduce damage to living body components are known (for example, Patent Document 5).
  • Patent Document 5 biocompatible materials that are used in contact with bodily fluids such as blood or living tissue and can reduce damage to living body components.
  • a biocompatible material is applied to a solid phase surface such as an immune reaction container or a measuring instrument, there may be a problem that the biocompatible material is removed from the solid phase surface in the washing step after the above-described adsorption treatment. .
  • the object of the present invention is to suppress, at a high level, nonspecific adsorption of a protein such as an antibody or an enzyme to the solid phase surface of a substrate such as an immune reaction container or a measurement instrument, and further improve the washing resistance. It is in providing the protein adsorption inhibitor which improves.
  • the present inventors have a specific range of lower critical shared temperature (hereinafter abbreviated as “LCST”) and a polyoxyethylene chain having a specific chain length (meth).
  • LCST lower critical shared temperature
  • meth a polyoxyethylene chain having a specific chain length
  • an acrylate copolymer can solve the above problems, and have completed the present invention. That is, the present invention includes the following [1] to [5].
  • [1] A water-soluble acrylic polymer containing a repeating structural unit [A] represented by the following formula (1), having a number average molecular weight of 5,000 to 250,000 and an LCST of 25 to 45 ° C.
  • [R 1 is a hydrogen atom or a methyl group
  • R 2 is a methyl group or an ethyl group
  • n is an average addition mole number of an oxyalkylene group of 2 to 3.
  • [2] A protein adsorption inhibitor coating solution containing the protein adsorption inhibitor of [1] above and water.
  • a base material provided with a coating layer of the protein adsorption inhibitor according to [1] on the surface.
  • a method for inhibiting protein adsorption comprising forming a coating layer on the surface of the substrate by P) and inhibiting adsorption of the protein to the substrate.
  • a water-soluble acrylic polymer (P) containing P) and having an LCST of 25 to 45 ° C. is supplied to the surface of the substrate to form a coating layer of the water-soluble acrylic polymer (P) on the surface of the substrate.
  • R 1 is a hydrogen atom or a methyl group
  • R 2 is a methyl group or an ethyl group
  • n is an average addition mole number of an oxyalkylene group of 2 to 3.
  • the protein adsorption inhibitor according to the present invention By removing the aqueous solution or the like in which the protein adsorption inhibitor according to the present invention is dissolved in contact with the solid surface of the substrate at a predetermined temperature, proteins such as antibodies and enzymes are non-specific on the solid surface of the substrate. Adsorption is suppressed at a high level. Furthermore, even when various washings are performed after removing the aqueous solution or the like by bringing it into contact with the solid phase surface of the base material, a protein adsorption inhibitor having improved washing resistance is provided.
  • the protein adsorption inhibitor of the present invention is a chemically synthesized product, the protein adsorption inhibitor containing it as an active ingredient is concerned with problems such as differences between lots or biological contamination of biological protein adsorption inhibitors. It exhibits protein adsorption inhibition ability safely and stably.
  • FIG. 1 is a diagram showing the protein adsorption inhibiting effect of a substrate provided with the coating layer of the protein adsorption inhibitor of Example 1 on the surface.
  • the protein adsorption inhibitor of the present invention contains a water-soluble acrylic polymer (P) as an active ingredient.
  • the water-soluble acrylic polymer (P) used in the present invention includes a repeating structural unit [A] represented by the following formula (1), and the repeating unit [A] is represented by the following formula (2). Obtained by polymerization of the monomer.
  • R 1 is a hydrogen atom or a methyl group, preferably a methyl group.
  • R 2 is a methyl group or an ethyl group, preferably a methyl group.
  • the water-soluble acrylic polymer (P) used in the present invention is selected (co) polymerized by selecting one or more of the monomers represented by the formula (2), and the LCST is 25 to 45 ° C. To be.
  • the LCST range is preferably 30 to 45 ° C, more preferably 33 to 43 ° C.
  • the LCST design of the copolymer can be estimated from the weighted average value of the homopolymer, and the required monomer blending ratio can be estimated.
  • LCST (lower critical shared temperature) in the present invention refers to a temperature at which the transmittance at a wavelength of 500 nm is 50% when a 1% by mass aqueous solution is heated at a heating rate of 1 ° C./min.
  • other comonomers can be used in combination.
  • intermediate water is a water molecule exhibiting a specific behavior, and is considered to exist in a material exhibiting biocompatibility according to recent research, for example, as a result of the following observation. For example, a differential scan of the endothermic amount observed when a sample containing water in PMEA (poly (2-methoxyethyl acrylate)) is cooled to ⁇ 100 ° C. and then heated at a rate of 2.5 ° C./min.
  • PMEA poly (2-methoxyethyl acrylate
  • a predetermined heat generation occurs in a specific temperature range of 0 ° C. or less (eg, around ⁇ 40 ° C.), and endotherm is observed in a wide temperature range from around ⁇ 10 ° C. to 0 ° C. Due to various studies, heat generation near ⁇ 40 ° C. is due to the regularization of some of the water molecules contained in PMEA, and the endotherm from around ⁇ 10 ° C. to 0 ° C. In this way, water molecules that exhibit behavior that does not occur in water alone are present in the water-containing PMEA, and this behavior is exhibited. Water is called “intermediate water” It is.
  • intermediate water is a biologically derived biocompatible material, such as biologically derived polysaccharides such as hyaluronic acid, heparin, gelatin, proteins such as albumin, nucleic acids such as DNA and RNA, and artificially synthesized materials. It is becoming clear that it is contained in substances having excellent biocompatibility, such as being contained in PEG. Thus, “intermediate water” is considered to be closely related to the development of biocompatibility in substances. The reason why “intermediate water” is generated in the substance is not clear, but it is the result of the interaction between the polymer chain with high molecular mobility in the substance and the water molecule with a specific intermolecular force. It is thought that there is. In biocompatible materials, the presence of intermediate water between the surface of the material and the hydration shell of the biological component prevents the direct contact between the two and suppresses foreign body reaction. .
  • LCST is a phase transition temperature similar to a so-called cloud point, and is a temperature at which a polymer such as polyalkylene glycol (meth) acrylate causes a coil-globule transition in an aqueous solution. That is, at a temperature lower than the LCST, the hydrophilicity of the polymer increases and is soluble in water, but the polymer under the temperature exceeding the LCST increases in hydrophobicity and becomes insoluble in water.
  • the polyalkylene glycol (meth) acrylate according to the present invention is a polymer that has been known to exhibit predetermined biocompatibility.
  • the polyalkylene glycol (meth) acrylate functions as a high-performance protein adsorption inhibitor by using a composition having a specific LCST value. That is, by removing an aqueous solution or the like in which the protein adsorption inhibitor according to the present invention is dissolved at a temperature equal to or lower than the LCST of the protein adsorption inhibitor to contact with the solid phase surface of the substrate to be treated, It was revealed that non-specific adsorption of proteins such as enzymes to the solid surface of the substrate can be suppressed at a high level.
  • the specific polyalkylene glycol (meth) acrylate of the present composition is water-soluble at the LCST temperature, but particularly in the temperature range immediately below the LCST. It has a driving force for precipitation, and when the aqueous solution comes into contact with the solid surface, the polymer precipitates in a form such as adsorption to the surface, and as a result, a coating that inhibits nonspecific adsorption of proteins is formed. It is thought to form. That is, according to the present invention, it is possible to realize an excellent protein adsorption inhibitor capable of preventing nonspecific adsorption of a protein to a substrate by a film formed of a polymer containing intermediate water.
  • the protein adsorption inhibitor is preferably a transparent and uniform solution when used. Because it is transparent, it does not interfere with optical detection even when a protein adsorption inhibitor is contained in the liquid to be evaluated. Also, since the protein adsorption inhibitor is a homogeneous solution, This is because the coating can be easily made uniform, that is, the thickness of the coating can be made uniform. On the other hand, when a protein adsorption inhibitor is used as an opaque solution, a cloudy solution enters the evaluation system, which may harm the detection and evaluation of the in vitro diagnostic agent in the optical system, and the resulting coating becomes non-uniform, and the ability to suppress protein adsorption may vary.
  • the protein adsorption inhibitor of the present invention contains a water-soluble acrylic polymer (P) having an LCST of 25 to 45 ° C. as an active ingredient, it can be easily transparent and uniform even when used at room temperature without heating. It is also excellent in that it can be.
  • P water-soluble acrylic polymer
  • the water-soluble acrylic polymer (P) used in the present invention has a number average molecular weight of 5,000 to 250,000, preferably 6,000 to 1,000,000, more preferably 7,000 to 200,000. It is. If the number average molecular weight is too low, the ability to suppress the adsorption of protein molecules will not be sufficient, and if it is too high, the water solubility will decrease, so the effects of the present invention may not be exhibited.
  • the value of the water-soluble acrylic polymer (P) polydispersity (Mw / Mn) is preferably 1.0 to 5.0, more preferably 1.0 to 4.4.
  • any of random copolymer, block copolymer, and graft copolymer may be used, and the copolymerization reaction for producing the copolymer is not particularly limited, and includes free radical polymerization, ionic polymerization, It can be used by a known synthesis method such as coordinate polymerization or ring-opening polymerization.
  • a water-soluble acrylic polymer (P) can be obtained by polymerizing a monomer in an inert gas atmosphere such as nitrogen gas or argon gas.
  • Examples of the organic solvent used in the polymerization of the monomer include alcohols such as methyl alcohol, ethyl alcohol, isopropyl alcohol, ethylene glycol, and propylene glycol; ketones such as acetone and methyl ethyl ketone; diethyl ether and tetrahydrofuran. Ethers such as benzene, toluene and xylene, and acetates such as methyl acetate and ethyl acetate, but are not limited thereto.
  • the concentration of the monomer in the monomer solution used in the solution polymerization method is not particularly limited, but is preferably about 10 to 80% by mass in consideration of the operability and efficiency of polymerization.
  • the polymerization initiator used above is not particularly limited.
  • azobisisobutyronitrile hereinafter abbreviated as “AIBN”
  • AIBN azobisisobutyronitrile
  • azoisobutyronitrile azoisobutyric acid methyl
  • azobisdimethylvaleronitrile peroxide
  • peroxide Light of azo polymerization initiators and peroxide polymerization initiators such as benzoyl, potassium persulfate and ammonium persulfate, benzophenone derivatives, phosphine oxide derivatives, benzoketone derivatives, phenylthioether derivatives, azide derivatives, diazo derivatives, disulfide derivatives, etc.
  • An initiator etc. are mentioned.
  • the amount of the polymerization initiator is not particularly limited, but usually it is preferably about 0.01 to 5 parts by mass with respect to 100 parts by mass of the monomer.
  • the polymer obtained by polymerization can be used after diluting and dissolving in an aqueous medium as it is, but it is preferable to carry out purification to remove impurities.
  • Known methods can be applied for purification. Specifically, a precipitation purification method in which a 10 to 80% by mass solution of a polymer in a good solvent is mixed with a 5 to 50 times poor solvent, and the good solvent solution of the polymer is compatible with the monomer.
  • an adsorption treatment method in which a monomer is separated by contacting with a certain adsorbent, and a membrane separation purification method of a monomer utilizing size separation by membrane separation of a good solvent solution of a polymer.
  • the protein adsorption inhibitor of the present invention is, for example, a protein, polypeptide, steroid, lipid, hormone, etc., more specifically, an enzyme reaction using various antigens, antibodies, receptors, enzymes, or the like, or an immunoglobulin antigen-antibody reaction It can be used in an immunological measurement method for measuring using Specifically, known radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), latex turbidimetry, etc., particularly preferably enzyme immunoassay (EIA), fluorescence immunity It can be applied to measurement methods (FIA), latex turbidimetry, Western blotting, etc.
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • FIA fluorescence immunoassay
  • EIA enzyme immunoassay
  • FSA fluorescence immunity
  • antibodies or antigens are bound to the solid phase surface, and then the antibody on the solid phase surface Alternatively, protein adsorption is suppressed by treating the solid surface to which no antigen is bound with the protein adsorption inhibitor of the present invention.
  • the solvent for dissolving the protein adsorption inhibitor for obtaining the protein adsorption inhibitor coating solution of the present invention is not only purified water, pure water, ion-exchanged water, but also a buffer that can be used in an immunological measurement method. Any liquid can be used. For example, phosphate buffer, acetate buffer, carbonate buffer, citrate buffer, Tris buffer, HEPES buffer, physiological saline, and the like can be used.
  • the protein adsorption inhibitor contained in the protein adsorption inhibitor coating solution is preferably 0.01% by mass or more, and more preferably 0.1% by mass or more.
  • the upper limit is not particularly limited as long as it dissolves in water as the main solvent, but is, for example, 20% by mass or less, preferably 10% by mass or less. This is because an effective protein adsorption suppressing effect can be obtained within these ranges.
  • a compound which can be contained in a protein adsorption inhibitor coating solution other than the protein adsorption inhibitor of the present invention the following compounds can be exemplified. That is, other reagents ordinarily used in this field, such as saccharides, salts, and surfactants. For example, as sugars, lactose, sucrose, trehalose and the like can be mentioned.
  • the salts include amino acids such as glycine, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, histidine and the like, peptides such as glycylglycine, phosphate, borate, sulfate
  • examples thereof include inorganic salts such as salts, tris salts, sodium chloride and potassium chloride, flavins, organic acids such as acetic acid, citric acid, malic acid, maleic acid and gluconic acid, and salts of organic acids.
  • the surfactant include polyoxyethylene sorbitan fatty acid ester and polyoxyethylene alkyl ether.
  • examples of compounds that can be contained in the coating solution of the protein adsorption inhibitor include water-soluble polymers such as MPC (2-methacryloyloxyethyl phosphorylcholine) polymer and polyvinyl alcohol.
  • the material of the base material used in the present invention is not particularly limited, but for example, polystyrene, polyvinyl chloride, polypropylene, acrylic resin, polymethyl methacrylate, polycarbonate, glass, metal, ceramic, silicon rubber, polyvinylidene fluoride, Examples thereof include nylon and nitrocellulose. Among these, polystyrene and polyvinylidene fluoride are preferable, and polystyrene is particularly preferable.
  • the shape of the substrate is not particularly limited, but specific examples include a film (film) shape, a plate shape, a particle shape, and a test tube shape, a vial shape, a flask shape, and the like. can do.
  • Examples of the method for forming a protein adsorption inhibitor coating layer on these substrates include the following methods. That is, the substrate is immersed in a protein adsorption inhibitor coating solution prepared by dissolving the protein adsorption inhibitor of the present invention in water or a solvent in which water and methanol, ethanol, and isopropanol are mixed at any ratio. Then, it can be dried at room temperature or by heating. Thereby, the coating layer is formed.
  • the concentration of the protein adsorption inhibitor is preferably 0.1 to 5.0% by mass, more preferably 1.0 to 3%. 0.0% by mass.
  • the solvent for diluting the protein adsorption inhibitor of the present invention is preferably water or a water / alcohol mixed solvent, more preferably water.
  • the protein adsorption inhibitor of the present invention As described above, by supplying a coating layer of the adsorption inhibitor on the substrate by supplying it to the surface of the substrate, the protein adsorption to the substrate during various measurements is performed.
  • a method of suppressing adsorption is one form of use. That is, the water-soluble acrylic polymer (P) used in the present invention is adsorbed as the coating layer on the solid phase surface of a substrate such as an immune reaction container or a measurement instrument, so that the protein is deposited on the solid phase surface. It suppresses adsorbing.
  • the reagent of the present invention is used in various measurements.
  • the method of adding a protein adsorption inhibitor is mentioned. That is, it is a method of forming a coating layer of the adsorption inhibitor on the substrate as one step of various measurements.
  • the protein adsorption inhibitor of this invention can also be added and used for all the reagents and solutions at the time of measuring.
  • the concentration of the protein adsorption inhibitor in each reagent and solution is preferably 0.0125 to 5.0% by mass, more preferably 0.1 to 5.0% by mass.
  • the protein adsorption inhibitor is added before adding a protein-containing sample such as serum, labeled antibody, or labeled antigen as the measurement target.
  • a protein or the like that can bind to a target component contained in a sample such as an antibody or an antigen is first bound to a fixed surface of a substrate such as the above-described immune reaction container or measurement instrument, and then the protein adsorption of the present invention is performed.
  • This is a method of treating with an inhibitor.
  • the protein binding inhibitor of the present invention is physically or chemically adsorbed to a protein that binds to the target component, for example, a substance that reacts with the measurement target, on the plate. It is the method of processing with.
  • the adsorption inhibitor of the present application is adsorbed to suppress adsorption of proteins to the plate surface on which the substance that binds to the measurement object is not adsorbed. It is a method to make it. As a result, a solid surface having a protein adsorption inhibiting effect can be obtained.
  • the base material of the same kind and shape as the above-mentioned "base material provided with a coating layer of a protein adsorption inhibitor on its surface" can be exemplified.
  • LCST lower critical shared temperature in the present invention refers to a temperature at which the transmittance at a wavelength of 500 nm is 50% when a 1% by mass aqueous solution is heated at a rate of temperature increase of 1 ° C./min. .
  • the LCST measurement was performed using a UV-visible spectrophotometer V-650 manufactured by JASCO Corporation.
  • ⁇ Synthesis Example 2> ⁇ Synthesis of ethoxytriethylene glycol monoacrylate homopolymer> The same procedure as in Synthesis Example 1 was performed except that ethoxytriethylene glycol monoacrylate was used as a raw material. It was confirmed by 1 H-NMR measurement that it was an ethoxytriethylene glycol monoacrylate homopolymer. The number average molecular weight (Mn) was 14,000 and the LCST was 33.8 ° C.
  • the number average molecular weight (Mn) was 7,000 and the LCST was 37.2 ° C.
  • ⁇ Synthesis Example 7> ⁇ Synthesis of methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (50/50, low molecular weight)> The same procedure as in Synthesis Example 1 was performed except that 3.5 g of methoxydiethylene glycol monomethacrylate and 4.0 g of methoxytriethylene glycol monoacrylate were used as raw materials, for a total of 7.5 g and 30 mg of initiator.
  • the number average molecular weight (Mn) was 17,000, and the LCST was 37.2 ° C.
  • the number average molecular weight (Mn) was 4,000 and the LCST was 52.0 ° C.
  • Example 1 ⁇ Preparation of protein adsorption inhibiting solution>
  • the polymer of Synthesis Example 1 was mixed in Dulbecco's phosphate buffer solution (manufactured by Sigma-Aldrich, hereinafter abbreviated as D-PBS) so as to be 0.1% by mass, and stirred and dissolved in a vortex mixer for 1 hour to adsorb protein. An inhibitor coating solution was obtained. The coating solution was transparent at room temperature, and it was considered that the whole amount of the charged polymer was dissolved.
  • D-PBS Dulbecco's phosphate buffer solution
  • the effect of the protein adsorption inhibitor according to the present invention will be described based on the schematic diagram of FIG.
  • a sample to be measured such as the antigen 14 is adsorbed to the antibody 16 on the left side of FIG.
  • the protein adsorption inhibitor 18 exerts an adsorption inhibiting effect, it is considered that adsorption of a specimen such as the antigen 14 is prevented like the antibody 16 on the right side of FIG.
  • the protein adsorption inhibitor of the present invention as shown on the right side of FIG. 1, adsorption of the specimen to the antibody 16 is effectively suppressed.
  • the washing resistance of the protein adsorption inhibitor coating solution was evaluated based on the “difference in protein adsorption rate” between “when not washed” and “after washing”. It can be said that the adsorption inhibitor having a small value of “difference in protein adsorption rate” ( ⁇ %) was well retained on the surface of the base material even after being washed, and can be said to exhibit good washing resistance.
  • Example 2-7 A protein adsorption inhibitor coating solution was prepared in the same manner as in Example 1 except that the polymer was used. Each coating solution was transparent at room temperature, and it was considered that the total amount of each charged polymer was dissolved. Furthermore, the protein adsorption inhibitory effect and washing resistance were evaluated in the same manner as in Example 1. The results are shown in Table 3.
  • Example 8> The polymer of Synthesis Example 4 was mixed with Dulbecco's phosphate buffer solution (manufactured by Sigma-Aldrich, hereinafter abbreviated as D-PBS) so as to be 0.04% by mass, and stirred and dissolved in a vortex mixer for 1 hour to adsorb protein. An inhibitor coating solution was obtained. The coating solution was transparent at room temperature, and it was considered that the whole amount of the charged polymer was dissolved. Otherwise, the evaluation was performed in the same manner as in Example 1.
  • D-PBS Dulbecco's phosphate buffer solution
  • a solution was prepared in the same manner as in Example 1 except that the compounds of Comparative Synthesis Examples 1 to 5 were used instead of the polymer of Synthesis Example 1.
  • the substrates treated with the protein adsorption inhibitor of the present invention were compared with the substrates treated with other oxyalkylene group-containing acrylic polymers (Comparative Examples 1 to 5). And has the ability to suppress nonspecific protein adsorption.
  • Such a result is that when the acrylic polymer (P) according to the present invention is brought into contact with the aqueous solution, a coating layer of the acrylic polymer (P) is formed on the surface, and protein adsorption is suppressed by the coating layer. It is considered a thing.
  • the protein adsorption inhibitor of each example has an overall "protein adsorption rate" value of "when not washed” and "after washing” as compared with the protein adsorption inhibitor of the comparative example. Since it was low, it was confirmed that the protein adsorption inhibitor of the present invention is excellent in nonspecific protein adsorption inhibiting ability. Furthermore, it was confirmed that the protein adsorption inhibitor of the present invention has excellent washing resistance. That is, in each Example of the present invention, the difference in protein adsorption rate from the unwashed case is also obtained after further washing after contacting the aqueous solution of the protein adsorption inhibitor with the substrate.
  • washing resistance ( ⁇ %) is small, which indicates that the adsorption inhibitor was well retained on the surface of the substrate even after washing.
  • the value of the washing resistance ( ⁇ %) was large, it was confirmed that the adsorption inhibitor was easily detached from the surface of the substrate by washing.
  • the substrate provided with the coating layers shown in Examples 4, 5, 7, and 8 on the surface had a protein adsorption inhibiting ability and washing resistance more predominately.
  • BSA whose results are shown in Table 4 is known to be excellent in protein adsorption inhibitor and washing resistance.
  • the protein adsorption inhibitors of the respective examples are substantially equivalent in adsorption inhibition effect and have higher durability cleaning properties.
  • the protein adsorption inhibitors of Examples 4, 5, 7, and 8 have better protein adsorption inhibition ability after washing than BSA as a reference example. As described above, it was confirmed that the stability of the analysis accuracy can be improved by using the protein adsorption inhibitor of the present invention.
  • Immune reaction container 12 Wall surface of the immune reaction container 14: Antigen (specimen) 16: Antibody 18: Protein adsorption inhibitor

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Coating Of Shaped Articles Made Of Macromolecular Substances (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)

Abstract

La présente invention aborde le problème de fourniture d'un inhibiteur d'adsorption de protéine qui peut inhiber l'adsorption non spécifique d'une protéine, par exemple un anticorps et une enzyme, sur une surface solide d'un matériau de base, par exemple, une cuve de réaction immunitaire et un outil de mesure, à un taux élevé et qui présente une résistance améliorée au frottement humide. Le problème peut être résolu par un inhibiteur d'adsorption de protéine qui contient un polymère acrylique soluble dans l'eau (P) en tant que substance active, le polymère acrylique soluble dans l'eau (P) contenant un motif constitutif de répétition [A] représenté par la formule (1), a un poids moléculaire moyen en nombre de 5 000 à 250 000 et a un LCST de 25 à 45°C. [R1 représente un atome d'hydrogène ou un groupe méthyle ; R2 représente un groupe méthyle ou un groupe éthyle ; et n représente le nombre moyen de moles d'un groupe oxyalkylène ajouté et représente 2 à 3.]
PCT/JP2017/009405 2016-03-10 2017-03-09 Inhibiteur d'adsorption de protéine et procédé d'inhibition d'adsorption de protéine Ceased WO2017155019A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2018504575A JP6803371B2 (ja) 2016-03-10 2017-03-09 蛋白質吸着抑制剤および蛋白質吸着抑制の方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2016-046829 2016-03-10
JP2016046829 2016-03-10

Publications (1)

Publication Number Publication Date
WO2017155019A1 true WO2017155019A1 (fr) 2017-09-14

Family

ID=59790614

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2017/009405 Ceased WO2017155019A1 (fr) 2016-03-10 2017-03-09 Inhibiteur d'adsorption de protéine et procédé d'inhibition d'adsorption de protéine

Country Status (2)

Country Link
JP (1) JP6803371B2 (fr)
WO (1) WO2017155019A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10153599A (ja) * 1996-11-22 1998-06-09 Nof Corp 蛋白質非特異的吸着防止剤およびその用途
JP3443891B2 (ja) * 1993-09-14 2003-09-08 日本油脂株式会社 タンパク質吸着防止剤
WO2008146631A1 (fr) * 2007-05-30 2008-12-04 Jsr Corporation Inhibiteur d'adsorption non spécifique
JP2011225769A (ja) * 2010-04-22 2011-11-10 Kawamura Institute Of Chemical Research 有機無機複合体分散液の製造方法
WO2013024815A2 (fr) * 2011-08-15 2013-02-21 一般財団法人川村理化学研究所 Copolymère séquencé et agent de revêtement antithrombotique
JP2013148484A (ja) * 2012-01-20 2013-08-01 Sumitomo Bakelite Co Ltd バイオチップの製造方法及びバイオチップ
JP2014102194A (ja) * 2012-11-21 2014-06-05 Sumitomo Bakelite Co Ltd 高分子物質、および高分子物質で表面が被覆された担体

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3443891B2 (ja) * 1993-09-14 2003-09-08 日本油脂株式会社 タンパク質吸着防止剤
JPH10153599A (ja) * 1996-11-22 1998-06-09 Nof Corp 蛋白質非特異的吸着防止剤およびその用途
WO2008146631A1 (fr) * 2007-05-30 2008-12-04 Jsr Corporation Inhibiteur d'adsorption non spécifique
JP2011225769A (ja) * 2010-04-22 2011-11-10 Kawamura Institute Of Chemical Research 有機無機複合体分散液の製造方法
WO2013024815A2 (fr) * 2011-08-15 2013-02-21 一般財団法人川村理化学研究所 Copolymère séquencé et agent de revêtement antithrombotique
JP2013057058A (ja) * 2011-08-15 2013-03-28 Kawamura Institute Of Chemical Research ブロック共重合体
JP2013148484A (ja) * 2012-01-20 2013-08-01 Sumitomo Bakelite Co Ltd バイオチップの製造方法及びバイオチップ
JP2014102194A (ja) * 2012-11-21 2014-06-05 Sumitomo Bakelite Co Ltd 高分子物質、および高分子物質で表面が被覆された担体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KITANO, HIROMI ET AL.: "Temperature-responsive polymer-brush constructed on a grass substrate by atom transfer radical polymerization", JOURNAL OF COLLOID AND INTERFACE SCIENCE, vol. 345, 2010, pages 325 - 331, XP055420902 *
YOON, JEONG AE ET AL.: "Comparison of the Thermoresponsive Deswelling Kinetics of Poly(2-(2-methoxyethoxy)ethyl methacrylate) Hydrogels Prepared by ATRP and FRP", MACROMOLECULES, vol. 43, 2010, pages 4791 - 4797, XP055420903 *

Also Published As

Publication number Publication date
JPWO2017155019A1 (ja) 2019-01-10
JP6803371B2 (ja) 2020-12-23

Similar Documents

Publication Publication Date Title
JP5344148B2 (ja) 非特異吸着防止剤および物品のコーティング方法
JP5365623B2 (ja) 生理活性物質の固定化方法
JP4733335B2 (ja) 再現性良好な凝集イムノアッセイ法及び試薬
CN107110841B (zh) 抑制蛋白质吸附的方法
JP2010117189A (ja) 生理活性物質固定化用基板
JP4821444B2 (ja) バイオアッセイ用高分子化合物およびこれを用いたバイオアッセイ用基材
US20150219641A1 (en) Analysis carrier and manufacturing method and use method therefor
JP2006176720A (ja) 医療材料用高分子化合物およびそれを用いた高分子溶液
JP6803371B2 (ja) 蛋白質吸着抑制剤および蛋白質吸着抑制の方法
JP6299862B2 (ja) コート剤組成物及びその利用
JP6085983B2 (ja) ブロッキング剤、標的物質に対する抗原または抗体が固定化された担体、これを含む体外診断用試薬およびキット、並びに標的物質の検出方法
JPWO2016039319A1 (ja) 蛋白質吸着抑制剤及び蛋白質吸着抑制方法
JP4984080B2 (ja) 免疫化学反応のシグナル増強剤および免疫学的測定方法
JP4640150B2 (ja) バイオチップおよびその使用方法
CN111094391B (zh) 聚乙二醇衍生物及蛋白质吸附抑制剂
JPWO2006123737A1 (ja) 医療材料用高分子化合物及び該高分子化合物を用いたバイオチップ用基板
WO2012015029A1 (fr) Réseau de chaînes de sucre
JP5958911B2 (ja) 糖ペプチドアレイ
JP4682828B2 (ja) バイオチップおよびその使用方法
JP5364971B2 (ja) 生理活性物質の固定化方法
JP5364973B2 (ja) 生理活性物質の固定化方法
JP2022158008A (ja) 体外診断用ブロッキング剤、これを含む体外診断用担体、およびキット
JP2017044705A (ja) 糖鎖アレイ
JP2015099162A (ja) 糖鎖アレイ

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 2018504575

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17763359

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17763359

Country of ref document: EP

Kind code of ref document: A1