JP6803371B2 - Protein adsorption inhibitor and method of protein adsorption inhibitor - Google Patents

Description

The present invention relates to a non-specific adsorption inhibitor for proteins and a method for suppressing adsorption. Specifically, a protein adsorption inhibitor that can prevent the adsorption of impurities (proteins) in a sample on the solid phase surface of a base material such as an immune reaction vessel or a measuring instrument and has high cleaning resistance, and a protein The present invention relates to a method for suppressing adsorption, and further to a substrate treated with the adsorption inhibitor.

For early detection of diseases, measurement methods using an immune response are widely used in the fields of clinical tests and diagnostic agents. Under these circumstances, higher sensitivity of tests is required, and improvement of sensitivity of clinical tests and diagnostic agents has become a major issue. In order to increase the sensitivity, the detection method is being switched from a method using an enzymatic reaction such as peroxidase or alkaline phosphatase to a method using fluorescence or chemiluminescence. It is said that the existence of one molecule of the substance to be inspected can be confirmed theoretically by using fluorescence or chemiluminescence as the detection method, but the desired sensitivity has not been obtained in practice.
As one of the factors that influence the detection sensitivity when measuring using an immune reaction, the antibody to be measured, the antigen, or the substrate of these labels used for measurement, such as an immune reaction container or a measuring instrument, is used. Non-specific adsorption to the solid phase surface can be mentioned. In addition, when a substance in which multiple types of biomolecules coexist, such as serum, plasma, cell extract, and urine, is used as a sample, an unspecified number of coexisting substances representing various proteins are used as a base material for an immune reaction vessel, a measuring instrument, or the like. The generation of noise due to non-specific adsorption on the solid surface of plasma is also a factor that hinders high sensitivity.

In order to prevent these non-specific adsorptions, as shown in Non-Patent Document 1 and Patent Document 1, biological proteins such as bovine serum albumin, casein, and gelatin, which are not conventionally involved in the immune response, are used as a buffer. A method of suppressing non-specific adsorption of proteins involved in an immune reaction by adsorbing biological proteins on the fixed surface of a base material such as an immune reaction vessel or a measuring instrument using a treatment solution as a solution. It is used. Furthermore, as a protein adsorption inhibitor containing a chemically synthesized product as a main component, Patent Document 2 contains 2-methacryloyloxyethyl phosphorylcholine polymer, Patent Document 3 contains glycosylethyl (meth) acrylate, and Patent Document 4 contains an oxyalkylene group. Each method using an acrylic polymer is disclosed. These methods are effective by physically adsorbing a chemically synthesized product as a protein adsorption inhibitor on the solid phase surface of an immune reaction vessel, a measuring instrument, or the like.

However, the adsorption inhibitory ability of the non-specific adsorption inhibitor based on the above-mentioned conventional technique may not be sufficient to suppress the generation of noise due to non-specific adsorption in the high-sensitivity measurement. There are many. This is not only because the function of the non-specific adsorption inhibitor is still insufficient, but also because the non-specific adsorption inhibitor can be easily used in the cleaning step performed using a buffer solution or an active agent aqueous solution after the non-specific adsorption treatment. This is also because the non-specific adsorption inhibitor for sufficiently exerting the non-specific adsorption inhibitory ability does not remain in the reaction vessel because of the desorption.

In addition, biocompatible materials that are used in contact with body fluids such as blood or biological tissues and can minimize damage to biological components are known (for example, Patent Document 5). However, even if such a biocompatible material is applied to the solid phase surface of an immune reaction vessel, a measuring instrument, or the like, there may be a problem that the biocompatible material is removed from the solid phase surface in the cleaning step after the adsorption treatment described above. ..

Johnson, D.A., Gautsch, J.W., Sportsman, J.R., and Elder, "Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose.", Gene Anal. Technol., 1, 3, 1984. 20.

Japanese Unexamined Patent Publication No. 07-270413 Japanese Unexamined Patent Publication No. 07-083923 Japanese Unexamined Patent Publication No. 10-123135 Japanese Unexamined Patent Publication No. 10-153599 International Publication No. 2004/087228 Pamphlet

As described above, the subject of the present invention is to suppress non-specific adsorption of proteins such as antibodies and enzymes to the solid phase surface of a substrate such as an immune reaction vessel or a measuring instrument at a high level, and further to improve cleaning resistance. The purpose is to provide a protein adsorption inhibitor to improve.

As a result of diligent studies in view of the above problems, the present inventors have a lower limit critical shared temperature in a specific range (hereinafter, abbreviated as "LCST") and a polyoxyethylene chain having a specific chain length (meth). We have found that an acrylate copolymer solves 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 the 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. A protein adsorption inhibitor containing P) as an active ingredient.

[R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, and n is the average number of moles of an oxyalkylene group added to 2-3. ]
[2] A protein adsorption inhibitor coating solution containing the protein adsorption inhibitor according to the above [1] and water.
[3] A base material provided with a coating layer of the protein adsorption inhibitor according to the above [1] on the surface.
[4] A water-soluble acrylic polymer containing the 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. A method for suppressing protein adsorption, in which a coating layer is formed on the surface of a base material according to P) to suppress the adsorption of proteins on the base material.

[R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, and n is the average number of moles of oxyalkylene groups added, which is 2-3. ]
[5] A water-soluble acrylic polymer containing the 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. A water-soluble acrylic polymer (P) containing P) and having an LCST of 25 to 45 ° C. is supplied to the surface of the base material to form a coating layer of the water-soluble acrylic polymer (P) on the surface of the base material. A method for imparting protein adsorption inhibitory properties to a substrate.

[R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, and n is the average number of moles of an oxyalkylene group added to 2-3. ]

[6] The above-mentioned [4], further comprising contacting a protein adsorption inhibitor containing the water-soluble acrylic polymer (P) with the surface of the substrate at a temperature equal to or lower than the LCST of the water-soluble acrylic polymer (P). The method for suppressing protein adsorption according to.

[7] The above-mentioned [5], further comprising contacting a protein adsorption inhibitor containing the water-soluble acrylic polymer (P) with the surface of the substrate at a temperature equal to or lower than the LCST of the water-soluble acrylic polymer (P). The method for imparting the protein adsorption inhibitory property according to.

By removing an aqueous solution or the like in which the protein adsorption inhibitor according to the present invention is dissolved by contacting it with the solid phase surface of the base material at a predetermined temperature, proteins such as antibodies and enzymes are nonspecific to the solid phase surface of the base material. Adsorption is suppressed at a high level. Further, even when various washings are performed after the aqueous solution or the like is brought into contact with the solid phase surface of the base material and removed, the effect is maintained, and a protein adsorption inhibitor having improved cleaning resistance is provided. Further, since the protein adsorption inhibitor of the present invention is a chemically synthesized product, the protein adsorption inhibitor containing it as an active ingredient may have problems such as lot difference and biological contamination of the biological protein adsorption inhibitor. It exhibits the ability to suppress protein adsorption safely and stably.

FIG. 1 is a diagram showing the protein adsorption inhibitory effect of a substrate provided with a coating layer of the protein adsorption inhibitor of Example 1 on the surface.

Hereinafter, the present invention will be described in more detail.
The protein adsorption inhibitor of the present invention contains a water-soluble acrylic polymer (P) as an active ingredient.
(Water-soluble acrylic polymer (P))
The water-soluble acrylic polymer (P) used in the present invention contains a repeating structural unit [A] represented by the following formula (1), and the repeating unit [A] is represented by the following formula (2). It is obtained by polymerization of the monomer.

In the formulas (1) and (2), R ¹ is a hydrogen atom or a methyl group, preferably a methyl group. Further, R ² is a methyl group or an ethyl group, preferably a methyl group. n is the average number of moles of the oxyalkylene group added, and m = 2 to 3, preferably 2.
Specific examples of the monomer represented by the formula (2) include methyldiethylene glycol acrylate (R ¹ = H, n = 2, R ² = CH ₃ ) and ethyltriethylene glycol acrylate (R ¹ = H, n). = 3, R ² = C ₂ H ₅ ), methyldiethylene glycol methacrylate (R ¹ = CH ₃ , n = 2, R ² = CH ₃ ), methyltriethylene glycol methacrylate (R ¹ = CH ₃ , n = 3, R) ² = CH ₃ ), methyltriethylene glycol acrylate (R ¹ = H, n = 3, R ² = CH ₃ ) and the like. Among these, methyl diethylene glycol methacrylate (R ¹ = CH ₃ , n = 2, R ² = CH ₃ ) is preferably mentioned.

The water-soluble acrylic polymer (P) used in the present invention is (co) polymerized by selecting one or more of the monomers represented by these formulas (2), and its LCST is set to 25 to 45 ° C. To be. The range of this LCST is preferably 30 to 45 ° C, more preferably 33 to 43 ° C. The LCST design of the copolymer can be predicted from the weighted average value of the homopolymer, and the required monomer compounding ratio can be estimated.
The LCST (lower limit critical shared temperature) in the present invention refers to the temperature at which the transmittance at a wavelength of 500 nm becomes 50% when the temperature of a 1% by mass aqueous solution is raised at a heating rate of 1 ° C./min.
Further, in the water-soluble acrylic polymer (P), other comonomer can be used in combination as a repeating unit other than the repeating structural unit [A].

It is generally known that polyalkylene glycol (meth) acrylate has intermediate water and is difficult to adsorb proteins. However, at the same time, the presence of intermediate water also affects the adsorption on the substrate.
The above-mentioned "intermediate water" is a water molecule that exhibits a peculiar behavior, and is considered to be present in a material exhibiting biocompatibility according to recent research, for example, based on the results of the following observations. For example, differential scanning shows 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 per minute. When measured with a calorimeter (DSC), a predetermined heat generation is generated in a specific temperature range below 0 ° C (for example, around -40 ° C), and endothermic is observed in a wide temperature range from around -10 ° C to 0 ° C. According to various studies, the heat generation at around -40 ° C is due to the regularization of some of the water molecules contained in the PMEA, and the endothermic at 0 ° C from around -10 ° C is due to the water molecules. It has been clarified that this is due to the irregularity again, and as described above, there are water molecules in the water-containing PMEA that exhibit behavior that does not occur with water alone, and exhibit such behavior. Water is called "intermediate water".
Such "intermediate water" is a biocompatible material such as hyaluronic acid derived from a living body, polysaccharides such as heparin, proteins such as gelatin and albumin, nucleic acids such as DNA and RNA, and artificially synthesized biocompatible 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 a substance is not clear, but it is the result of the interaction between a polymer chain with high molecular mobility in a substance and water molecules with a specific intermolecular force. It is believed that there is. In biocompatible materials, it is considered that the presence of intermediate water between the surface of the material and the hydrated shell of the biological component prevents direct contact between the two materials and suppresses the foreign body reaction of the living body. ..

Further, 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 below LCST, the hydrophilicity of the polymer increases and it is soluble in water, but at a temperature above LCST, the polymer becomes more hydrophobic and becomes insoluble in water.
The polyalkylene glycol (meth) acrylate according to the present invention is a polymer known to exhibit predetermined biocompatibility. On the other hand, it has been clarified by the present invention that the polyalkylene glycol (meth) acrylate unexpectedly functions as a high-performance protein adsorption inhibitor by using a composition having a specific LCST value. That is, an antibody or an antibody or the like is removed by contacting an aqueous solution or the like in which the protein adsorption inhibitor according to the present invention is dissolved with the solid surface of the substrate to be treated at a temperature equal to or lower than LCST of the protein adsorption inhibitor. It has been clarified that it is possible to suppress non-specific adsorption of proteins such as enzymes on the solid surface of the substrate at a high level. Further, since the effect is maintained even when various cleanings are performed after the aqueous solution or the like is brought into contact with the solid phase surface of the base material and removed, various cleanings are performed when actually performing various evaluations. Even in this case, non-specific adsorption of protein can be suppressed. Although the mechanism of action that causes such a phenomenon has not been elucidated, the specific polyalkylene glycol (meth) acrylate of the present application configuration is water-soluble under the temperature of LCST, but especially in the temperature range immediately below LCST. It has a driving force for precipitation, and when an aqueous solution comes into contact with a solid surface, the polymer precipitates in the form of adsorption on the surface, and as a result, a film that inhibits non-specific 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 non-specific adsorption of a protein on a substrate by a coating film formed of a polymer containing intermediate water.

Further, the protein adsorption inhibitor is preferably a transparent and uniform solution at the time of use. Being transparent, it does not interfere with optical detection even when the liquid to be evaluated contains a protein adsorption inhibitor, and because the protein adsorption inhibitor is a uniform solution, the protein adsorption inhibitor 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 turbid solution may enter the evaluation system and impair the detection and evaluation of the in vitro diagnostic agent in the optical system, and the coating produced. Is non-uniform, and the performance of suppressing protein adsorption may vary.
Since 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 is easily a transparent and uniform solution even when used at room temperature without heating. It is also excellent in that it can be used.

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, and more preferably 7,000 to 200,000. Is. If the number average molecular weight is too low, the ability to suppress the adsorption of protein molecules is insufficient, and if it is too high, the water solubility is lowered, so that the effect of the present invention may not be exhibited.

In the water-soluble acrylic polymer (P) used in the present invention, by aligning the molecular weight distribution, it is possible to eliminate the variation between lots when used in the field of diagnostic agents. Therefore, the value of the water-soluble acrylic polymer (P) polydispersity (Mw / Mn) is preferably 1.0 to 5.0, and more preferably 1.0 to 4.4. Further, a random copolymer, a block copolymer, or a graft copolymer may be used, and the copolymerization reaction itself for producing the copolymer is not particularly limited, and free radical polymerization, ionic polymerization, etc. It can be used by known synthetic methods such as distribution polymerization and ring-opening polymerization. For example, it can be prepared by polymerizing a monomer by a solution polymerization method using water or an organic solvent as a solvent. More specifically, one or more monomers described in the formula (1) are dissolved in purified water or an organic solvent, and the obtained solution is stirred while a polymerization initiator is added to the solution. A water-soluble acrylic polymer (P) can be obtained by polymerizing the monomer in an inert gas atmosphere such as nitrogen gas or argon gas.

Examples of the organic solvent used in the polymerization of the above-mentioned monomers 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, xylene and the like, aromatic compounds such as methyl acetate and ethyl acetate and the like, 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 operability and efficiency of polymerization.
The polymerization initiator used above is not particularly limited, but is, for example, azobisisobutyronitrile (hereinafter abbreviated as "AIBN"), azoisobutyronityl, methyl azoisobutyrate, azobisdimethylvaleronitrile, peroxide. Light from azo-based polymerization initiators such as benzoyl, potassium persulfate, ammonium persulfate, peroxide-based polymerization initiators, benzophenone derivatives, phosphine oxide derivatives, benzoketone derivatives, phenylthioether derivatives, azide derivatives, diazo derivatives, disulfide derivatives, etc. Initiators and the like can be mentioned. The amount of the polymerization initiator is not particularly limited, but is usually preferably about 0.01 to 5 parts by mass with respect to 100 parts by mass of the monomer.
The polymer obtained by the polymerization can be used as it is by diluting and dissolving it in an aqueous medium, but it is preferable to purify it in order 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-fold poor solvent, and a good solvent solution of the polymer has an affinity with a monomer. Examples thereof include an adsorption treatment method in which a polymer is brought into contact with a certain adsorbent to separate the monomer, and a membrane separation and purification method for a monomer using size separation by membrane separation of a good solvent solution of a polymer.

The protein adsorption inhibitor of the present invention is, for example, an enzyme reaction using proteins, polypeptides, steroids, lipids, hormones, etc., more specifically, various antigens, antibodies, receptors, enzymes, etc., or an immunoglobulin antigen-antibody reaction. It can be used in an immunological measurement method or the like for measuring using. Specifically, known radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), latex turbidimetric method and the like, particularly preferably enzyme immunoassay (EIA), fluorescence immunoassay. It can be applied to measurement methods (FIA), latex turbidity methods, western blotting, etc. In these known immunoassays, after binding an antibody or antigen to the solid phase surface, the antibody on the solid phase surface Alternatively, the solid phase surface to which the antigen is not bound is treated with the protein adsorption inhibitor of the present invention to suppress protein adsorption.

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, and ion-exchanged water, but also a buffer that can be used in an immunological measurement method. Any liquid can be used. For example, a phosphate buffer solution, an acetate buffer solution, a carbonate buffer solution, a citrate buffer solution, a Tris buffer solution, a HEPES buffer solution, a physiological saline solution and the like can be used.
Further, the protein adsorption inhibitor contained in the protein adsorption inhibitor coating liquid 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 is dissolved 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 inhibitory effect can be obtained within these ranges.

In addition to the protein adsorption inhibitor of the present invention, the following compounds can be exemplified as compounds that can be contained in the protein adsorption inhibitor coating solution.
That is, other reagents and the like usually used in this field, and examples thereof include saccharides, salts, and surfactants. For example, examples of sugars include lactose, sucrose, trehalose and the like. For example, as salts, amino acids and amino acid salts such as glycine, alanine, serine, threonine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, histidine, peptides such as glycylglycine, phosphates, borates, sulfate Examples thereof include inorganic salts such as salts, tris salts, sodium chloride and potassium chloride, organic acids such as flavins, acetic acid, citric acid, malic acid, maleic acid and gluconic acid, and salts of organic acids. For example, examples of the surfactant include polyoxyethylene sorbitan fatty acid ester and polyoxyethylene alkyl ether.
Further, examples of the compound that can be contained in the coating liquid of the protein adsorption inhibitor include water-soluble polymers such as MPC (2-methacryloyloxyethyl phosphorylcholine) polymer and polyvinyl alcohol.

Next, a base material having a coating layer of a protein adsorption inhibitor on the surface will be described.
The material of the base material used in the present invention is not particularly limited, but for example, polystyrene, polyvinyl chloride, polypropylene, acrylic resin, polymethylmethacrylate, polycarbonate, glass, metal, ceramic, silicon rubber, polyvinylidene fluoride, etc. Nylon, nitrocellulose and the like can be mentioned. Among them, polystyrene and polyvinylidene fluoride are preferable, and polystyrene is particularly preferable.
The shape of the base material is not particularly limited, but specifically, a film shape, a plate shape, a particle shape, a test tube shape, a vial bottle shape, a flask shape, and the like are exemplified. can do.

Examples of the method for forming the coating layer of the protein adsorption inhibitor 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 obtained by mixing water with methanol, ethanol, and isopropanol in an arbitrary ratio thereof. It only needs to be dried, and then it can be dried at room temperature or by heating. As a result, the coating layer is formed.
In the protein adsorption inhibitor coating solution for forming a coating layer of the protein adsorption inhibitor, the concentration of the protein adsorption inhibitor is preferably 0.1 to 5.0% by mass, more preferably 1.0 to 3%. It is 0.0% by mass. Further, as the solvent for diluting the protein adsorption inhibitor of the present invention, water and a water / alcohol mixed solvent are preferable, and water is more preferable.

Next, a method of using the protein adsorption inhibitor of the present invention will be described.
In the protein adsorption inhibitor of the present invention, as described above, by supplying the protein to the surface of the substrate to form a coating layer of the adsorption inhibitor on the substrate, the protein on the substrate at the time of various measurements can be measured. A method of suppressing adsorption is one form of use. That is, when the water-soluble acrylic polymer (P) used in the present invention is adsorbed as the coating layer on the solid phase surface of a base material such as an immune reaction vessel or a measuring instrument, a protein is formed on the solid phase surface. It suppresses adsorption.
In addition to the method of preparing a base material having a coating layer of a protein adsorption inhibitor on the surface in advance and using the base material for an immune reaction vessel, a measuring instrument, etc., the reagent used in various measurements of the present invention is used. Examples thereof include a method of adding a protein adsorption inhibitor. That is, as one step of various measurements, it is a method of forming a coating layer of the adsorption inhibitor on the base material. It is also possible to add the protein adsorption inhibitor of the present invention to all the reagents and solutions for carrying out the measurement.
In such a method of use, 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. .. However, when a protein adsorption inhibitor is added as one step of various measurements, the protein adsorption inhibitor is added before adding a protein-containing sample such as serum, a labeled antibody or a labeled antigen, which is a measurement target.

Further, as another usage pattern, the following method can be mentioned.
That is, the protein of the present invention is adsorbed after first binding a protein or the like that can bind to a target component contained in a sample such as an antibody or an antigen to the fixed surface of a base material such as the immune reaction vessel or the measuring instrument. This is a method of treating with an inhibitor. For example, when a polystyrene plate is used, a protein that binds to a target component, for example, a substance that reacts with an object to be measured is physically or chemically adsorbed on the plate, and then the protein adsorption inhibitor of the present invention is used. It is a method of processing with. That is, after adsorbing a substance that can bind to the measurement target on the plate surface, the adsorption inhibitor of the present application is adsorbed in order to suppress the adsorption of the protein on the plate surface on which the substance that binds to the measurement target is not adsorbed. It is a method to make it. As a result, a solid phase surface having a protein adsorption inhibitory effect can be obtained.
As the base material in such a usage method, a base material having the same type and shape as the above-mentioned "base material having a coating layer of a protein adsorption inhibitor on the surface" can be exemplified.

<Synthesis example 1>
<Synthesis of methoxydiethylene glycol monoacrylate homopolymer>
15 g of methoxydiethylene glycol monoacrylate as a monomer was weighed in a four-necked flask, 15 mg of azobisisobutyronitrile as a radical polymerization initiator was added, and the mixture was dissolved in 60 g of 1,4-dioxane at room temperature. After confirming the dissolution of the initiator, the temperature was raised to 75 ° C. in an oil bath, and after reaching 75 ° C., the polymerization reaction was carried out for 8 hours. After completion of the polymerization reaction, 1,000 mL of the reaction solution was diluted with about 1,000 mL of hexane and washed to remove the organic phase to recover the product, which was redissolved in 70 mL of tetrahydrofuran and washed again with n-hexane. The product was recovered and dried under reduced pressure for 24 hours to obtain the desired oxyalkylene polymer. ¹ It was confirmed by ¹ H-NMR measurement that the product was a methoxydiethylene glycol monoacrylate homopolymer. Moreover, as a result of the molecular weight analysis of GPC, the number average molecular weight (Mn) was 13,000. Further, an aqueous solution containing 1% by mass of the polymer was prepared, and it was confirmed by UV measurement at a wavelength of 500 nm that the LCST of the polymer in the aqueous solution was 43.1 ° C.
As described above, the LCST (lower limit critical point shared temperature) in the present invention refers to the temperature at which the transmittance at a wavelength of 500 nm becomes 50% when the temperature of a 1 mass% aqueous solution is raised at a temperature rise rate of 1 ° C./min. .. For the measurement of LCST, an ultraviolet-visible spectrophotometer V-650 manufactured by JASCO Corporation was used. The measurement was performed based on the value of the transmittance of the aqueous solution at that time.
In the water-soluble acrylic polymer (P) used in the present invention, one or more of these monomers represented by the formula (2) are selected and (co) polymerized, and the LCST thereof is 25 to 45 ° C. To be. The range of this LCST is preferably 30 to 45 ° C, more preferably 33 to 43 ° C. The LCST design of the copolymer can be predicted from the weighted average value of the homopolymer, and the required monomer compounding ratio can be estimated.

<Synthesis example 2>
<Synthesis of ethoxytriethylene glycol monoacrylate homopolymer>
The procedure was the same as in Synthesis Example 1 except that ethoxytriethylene glycol monoacrylate was used as a raw material. ¹ 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.

<Synthesis example 3>
<Synthesis of methoxytriethylene glycol monomethacrylate homopolymer>
The same procedure as in Synthesis Example 1 was carried out except that methoxytriethylene glycol monomethacrylate was used as a raw material. ¹ It was confirmed by ¹ H-NMR measurement that it was a methoxytriethylene glycol monomethacrylate homopolymer. The number average molecular weight (Mn) was 105,000 and the LCST was 42.5 ° C.

<Synthesis example 4>
<Synthesis of methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (50/50)>
The same procedure as in Synthesis Example 1 was carried out except that 6.9 g of methoxydiethylene glycol monomethacrylate and 8.1 g of methoxytriethylene glycol monoacrylate were used as raw materials for a total of 15 g. ¹ Confirmation by H-NMR measurement that the product is a methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (methoxydiethylene glycol monomethacrylate / methoxytriethylene glycol monoacrylate = 47/53; molar ratio). did. The number average molecular weight (Mn) was 48,000 and the LCST was 37.2 ° C.

<Synthesis example 5>
<Synthesis of methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monomethacrylate copolymer (60/40)>
The same procedure as in Synthesis Example 1 was carried out except that 8.2 g of methoxydiethylene glycol monomethacrylate and 6.8 g of methoxytriethylene glycol monomethacrylate were used as raw materials for a total of 15 g. ¹ Confirmation by H-NMR measurement that the product is a methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monomethacrylate copolymer (methoxydiethylene glycol monomethacrylate / methoxytriethylene glycol monomethacrylate = 58/42; molar ratio). did. The number average molecular weight (Mn) was 188,000 and the LCST was 35.1 ° C.

<Synthesis example 6>
<Synthesis of methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (40/60)>
The same procedure as in Synthesis Example 1 was carried out except that 5.5 g of methoxydiethylene glycol monomethacrylate and 9.5 g of methoxytriethylene glycol monoacrylate were used as raw materials for a total of 15 g. ^{1 By} H-NMR measurement, the product is a methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (methoxydiethylene glycol monomethacrylate / methoxytriethylene glycol monoacrylate copolymer = 35/65; molar ratio). It was confirmed. 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)>
As raw materials, 3.5 g of methoxydiethylene glycol monomethacrylate and 4.0 g of methoxytriethylene glycol monoacrylate were added to a total of 7.5 g, and the initiator was 30 mg, and the same procedure as in Synthesis Example 1 was carried out. ^{1 By} H-NMR measurement, the product is a methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (methoxydiethylene glycol monomethacrylate / methoxytriethylene glycol monoacrylate copolymer = 47/53; molar ratio). It was confirmed. The number average molecular weight (Mn) was 17,000 and the LCST was 37.2 ° C.

<Comparative synthesis example 1>
<Synthesis of methoxytriethylene glycol monoacrylate homopolymer>
The procedure was the same as in Synthesis Example 1 except that methoxytriethylene glycol monomethacrylate was used as a raw material. ¹ It was confirmed by ¹ H-NMR measurement that the product was a methoxytriethylene glycol monoacrylate copolymer. The number average molecular weight (Mn) was 12,000 and the LCST was 63.8 ° C.

<Comparative synthesis example 2>
<Synthesis of 2-methoxyethyl vinyl ether homopolymer>
15 g of 2-methoxyethyl vinyl ether monomer as a raw material is diluted with 15 g of dry ethyl acetate, weighed in a four-necked flask, 15 mL of a toluene solution of 1-butoxyethyl acetate 40 mM as a polymerization initiator is added, and argon is added in 180 mL of dry toluene. After cooling to 0 ° C. under an air stream, 3.75 mL of a 1 M toluene solution of Et _1.5 AlCl _1.5 was added as Lewis acid with stirring. After stirring this at 0 ° C. for 30 minutes, 22.5 mL of ethanol was added to the reaction solution to stop the reaction. After completion of the polymerization reaction, the reaction solution was diluted with toluene and washed, and the solvent was distilled off from the organic phase to obtain a product. Then, washing was performed three times with warm water at 80 ° C. The product was recovered and dried under reduced pressure for 24 hours to obtain the desired oxyalkylene polymer. The structure of the obtained polymer was confirmed to be a 2-methoxyethyl vinyl ether homopolymer by ¹ H-NMR measurement. The number average molecular weight (Mn) was 26,000 and the LCST was 69.0 ° C.

<Comparative synthesis example 3>
<Synthesis of methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (20/80, low molecular weight)>
The same procedure as in Synthesis Example 1 was carried out except that 1.3 g of methoxydiethylene glycol monomethacrylate and 6.2 g of methoxytriethylene glycol monoacrylate were used as raw materials to make a total of 7.5 g, and the initiator was 30 mg. ^{1 By} H-NMR measurement, the product is a methoxydiethylene glycol monomethacrylate-methoxytriethylene glycol monoacrylate copolymer (methoxydiethylene glycol monomethacrylate / methoxytriethylene glycol monoacrylate copolymer = 15/85; molar ratio). It was confirmed. The number average molecular weight (Mn) was 4,000 and the LCST was 52.0 ° C.

<Comparative synthesis example 4>
<Synthesis of methoxytetraethylene glycol monoacrylate homopolymer>
The procedure was the same as in Synthesis Example 1 except that methoxytetraethylene glycol monoacrylate was used as a raw material. ¹ It was confirmed by ¹ H-NMR measurement that the product was a methoxytetraethylene glycol monoacrylate homopolymer. The number average molecular weight (Mn) was 87,000, and the LCST was 75 ° C. or higher.

<Comparative synthesis example 5>
<Synthesis of methoxytetraethylene glycol monomethacrylate homopolymer>
The procedure was the same as in Synthesis Example 1 except that methoxytetraethylene glycol monomethacrylate was used as a raw material. ¹ It was confirmed by ¹ H-NMR measurement that the product was a methoxytetraethylene glycol monomethacrylate homopolymer. The number average molecular weight (Mn) was 122,000, and the LCST was 75 ° C. or higher.
<Comparative synthesis example 6>
<Synthesis of methoxydiethylene glycol monomethacrylate homopolymer>
The procedure was the same as in Synthesis Example 1 except that methoxydiethylene glycol monomethacrylate was used as a raw material. ¹ It was confirmed by ¹ H-NMR measurement that the product was a methoxydiethylene glycol monomethacrylate homopolymer. The number average molecular weight (Mn) was 25,000 and the LCST was 23.5 ° C.

The meanings of the abbreviations in the table are as follows.
* Mn: Number average molecular weight * * LCST: Lower Critical Solution Temperature

<Example 1>
<Preparation of protein adsorption suppression solution>
The polymer of Synthesis Example 1 is mixed with a darbecolinic acid buffer solution (manufactured by Sigma-Aldrich, hereinafter abbreviated as D-PBS) so as to have a content of 0.1% by mass, dissolved by stirring with a vortex mixer for 1 hour, and protein adsorption. It was used as an inhibitor coating solution. The coating liquid was transparent at room temperature, and it was considered that the entire amount of the polymer charged was dissolved.

<Evaluation of protein adsorption inhibitory effect>
The protein adsorption inhibitory effect of the protein adsorption inhibitor coating solution of Example 1 was measured by the following method.
A protein adsorption inhibitor coating solution was dispensed at 200 μL / well onto a MaxiSoap plate (polystyrene plate manufactured by Thermo Fisher Scientific), and allowed to stand at room temperature for 2 hours. After that, the solution was completely removed with an ejector, and one of them was left as it was (the result is described in the column labeled "Unwashed" in Table 3), and the other immediately dispensed D-PBS to 200 μL / well. The operation of removing the liquid with the aspirator was repeated 5 times each (the results are described in the column labeled "after cleaning" in Table 3). Subsequently, POD-IgG (peroxidase-labeled immunoglobulin G, manufactured by Biorad) diluted 20,000 times with D-PBS was dispensed into 100 μL / well and allowed to stand at room temperature for 2 hours. Then, D-PBS containing Tween 20 at a concentration of 0.05% was dispensed into 200 μL / well, and the solution was completely removed with an ejector. This operation was repeated 4 times to clean the plate surface. After washing, 100 μL / well of a color-developing solution prepared using TMB Microwellell Peroxidase Substrate (manufactured by KPL) was added, and the mixture was reacted at room temperature for 7 minutes. The color development reaction was stopped by dispensing a 1 mol / L sulfuric acid solution to 50 μL / well, and the absorbance at 450 nm was measured to detect the adsorbed protein. The smaller the absorbance, the more the protein adsorption is suppressed. A Spectra Max M3 (manufactured by Molecular Device) was used for the absorbance measurement. The evaluation results are shown in Table 3.

The effect of the protein adsorption inhibitor according to the present invention will be described with reference to the schematic diagram of FIG. For example, when the wall surface 12 of the immune reaction vessel 10 is used as a base material, on the left side of FIG. 1, a sample to be measured such as an antigen 14 is adsorbed on the antibody 16. On the other hand, when the protein adsorption inhibitor 18 exerts an adsorption inhibitory effect, it is considered that the adsorption of a sample such as an antigen 14 is prevented as shown by the antibody 16 on the right side of FIG. As will be described in detail later, according to the protein adsorption inhibitor of the present invention, the adsorption of the sample to the antibody 16 is effectively suppressed as shown on the right side of FIG.

<Evaluation of cleaning resistance>
The cleaning resistance of the protein adsorption inhibitor coating solution was evaluated based on the "difference in protein adsorption rate" between the above "unwashed" and the above "after cleaning". It can be said that the adsorption inhibitor having a small value of "difference in protein adsorption rate" (Δ%) was well retained on the surface of the base material even after being washed, and can be said to exhibit good washing resistance.

<Examples 2 to 7>
A protein adsorption inhibitor coating solution was prepared in the same manner as in Example 1 except that the polymers of Synthesis Examples 2 to 7 were used. Each coating liquid was transparent at room temperature, and it was considered that the entire amount of the polymer charged was dissolved. Further, the protein adsorption inhibitory effect and the cleaning 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 a darbeckolinic acid buffer solution (manufactured by Sigma-Aldrich, hereinafter abbreviated as D-PBS) so as to be 0.04% by mass, dissolved by stirring with a vortex mixer for 1 hour, and protein adsorption. It was used as an inhibitor coating solution. The coating liquid was transparent at room temperature, and it was considered that the entire amount of the polymer charged was dissolved. Other than that, the evaluation was carried out in the same manner as in Example 1.
<Comparative Examples 1 to 5>
The 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. Each solution was transparent at room temperature, and it was considered that the entire amount of the polymer charged was dissolved. Further, the protein adsorption inhibitory effect and the cleaning resistance were evaluated in the same manner as in Example 1. The results are shown in Table 4.
<Comparative Example 6>
The compound of Comparative Synthesis Example 6 was used instead of the polymer of Synthesis Example 1, but the protein adsorption inhibitory effect and the washing resistance test were stopped because turbidity occurred.
A protein adsorption-suppressing solution was prepared in the same manner as in Example 1 except that a protein (BSA) derived from albumin bovine serum was used instead of the polymer of Synthesis Example 1. Further, the protein adsorption inhibitory effect and the cleaning resistance were evaluated in the same manner as in Example 1. The results are shown in Table 4.

The base materials treated with the protein adsorption inhibitor of the present invention (Examples 1 to 8) were compared with the base materials treated with other oxyalkylene group-containing acrylic polymers (Comparative Examples 1 to 5). It had the ability to suppress non-specific protein adsorption. As a result, by contacting with the aqueous solution of the acrylic polymer (P) according to the present invention, a coating layer of the acrylic polymer (P) was formed on the surface, and protein adsorption was suppressed by this coating layer. It is considered to be.
That is, the protein adsorption inhibitor of each example generally has a higher value of "protein adsorption rate" in both "unwashed" and "after washing" than the protein adsorption inhibitor of the comparative example. Since it was low, it was confirmed that the protein adsorption inhibitor of the present invention was excellent in non-specific protein adsorption inhibitory ability.
Furthermore, it was confirmed that the protein adsorption inhibitor of the present invention has excellent cleaning resistance. That is, in each of the examples of the present invention, even when the aqueous solution of each protein adsorption inhibitor is removed by contacting the base material and then further washed, the difference in the protein adsorption rate from that in the unwashed state is observed. The value of the corresponding cleaning resistance (Δ%) was small, which indicates that the adsorption inhibitor was well retained on the surface of the substrate even after cleaning. On the other hand, in each of the comparative examples, the value of the cleaning resistance (Δ%) was large, so it was confirmed that the adsorption inhibitor was easily removed from the surface of the base material by cleaning. In particular, the base material provided with the coating layer shown in Examples 4, 5, 7, and 8 on the surface had a protein adsorption suppressing ability and cleaning resistance more predominantly.
As a reference comparative example, BSA whose results are shown in Table 4 is known to be excellent in protein adsorption inhibitor and cleaning resistance. Compared with such a reference comparative example, it can be seen that the protein adsorption inhibitor of each example has almost the same adsorption inhibitory effect and has higher durable detergency. In particular, the protein adsorption inhibitors of Examples 4, 5, 7, and 8 are more excellent in the ability to suppress protein adsorption after washing than BSA, which is a reference example. As described above, it was confirmed that the stability of analysis accuracy can be improved by using the protein adsorption inhibitor of the present invention.

10: Immune reaction container 12: Wall surface of immune reaction container 14: Antigen (specimen)
16: Antibody 18: Protein adsorption inhibitor

Claims (9)

A water-soluble acrylic polymer (P) containing the 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 30 to 45 ° C. A protein adsorption inhibitor contained as an active ingredient.
[R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, and n is the average number of moles of an oxyalkylene group added to 2-3. ] A protein adsorption inhibitor coating solution containing the protein adsorption inhibitor according to claim 1 and water. The protein adsorption inhibitor coating solution according to claim 2, wherein the concentration of the protein adsorption inhibitor is 0.1 to 5.0% by mass. A base material provided with a coating layer of the protein adsorption inhibitor according to claim 1 on the surface. The base material according to claim 4, wherein the coating layer contains a water-soluble acrylic polymer (P) adsorbed on the solid phase surface of the base material. By a water-soluble acrylic polymer (P) containing the 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 30 to 45 ° C. A method for suppressing protein adsorption, wherein a coating layer is formed on the surface of the base material to suppress the adsorption of proteins on the base material.
[R ¹ is a hydrogen atom or a methyl group, R ² is a methyl group or an ethyl group, and n is the average number of moles of an oxyalkylene group added to 2-3. ] A water-soluble acrylic polymer (P) 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 30 to 45 ° C. , A method of imparting protein adsorption inhibitory property to a substrate by supplying the substrate to the surface of the substrate and forming a coating layer of a 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, and n is the average number of moles of an oxyalkylene group added to 2-3. ] The sixth aspect of claim 6 , further comprising contacting the protein adsorption inhibitor containing the water-soluble acrylic polymer (P) with the surface of the base material at a temperature equal to or lower than the LCST of the water-soluble acrylic polymer (P). Method of suppressing protein adsorption. The seventh aspect of claim 7 , further comprising bringing the protein adsorption inhibitor containing the water-soluble acrylic polymer (P) into contact with the surface of the base material at a temperature equal to or lower than the LCST of the water-soluble acrylic polymer (P). A method for imparting protein adsorption inhibitory properties.