Blocking agent, kit and blocking method

Abstract

To provide a blocking agent excellent in a non-specific adsorption preventing ability, a protein stabilizing ability, and an effect of color developing sensitization generated in an immunostaining procedure; and to provide a kit and a blocking method.SOLUTION: A blocking agent for preventing non-specific adsorption in antigen-antibody reaction carried out on a planar base material, comprises a water-soluble vinyl-based polymer (A), the water-soluble vinyl-based polymer (A) comprising a structural unit (a), a structural unit (b) and a structural unit (c). In the blocking agent, the content of the structural unit (c) is 5 to 35 mass% in 100 mass% of the water-soluble vinyl-based polymer (A). The structural unit (a) is a structural unit having a zwitterionic structure; the structural unit (b) is a structural unit derived from a monomer (b') with a distribution coefficient LogP of 0.8 to 10; and the structural unit (c) is a structural unit (where, excluding any having a zwitterionic structure) derived from a monomer (c') with a pK of -3 to 5 in water.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a blocking agent, a kit, and a blocking method. [Background technology]

[0002] In the field of biochemical analysis, Western blotting, ELISA, immunostaining, and the like are widely used as methods for detecting and visualizing proteins by utilizing specific adsorption due to antigen-antibody reactions. It is generally known that antibodies cause non-specific adsorption reactions with proteins other than the target protein. In order to prevent such non-specific adsorption, a pretreatment is performed in which the surface to be detected or measured is covered with a blocking agent that does not interfere with specific adsorption. In the immunostaining method, normal serum, bovine serum albumin (BSA), gelatin, skim milk, and the like are used as blocking agents. (Patent Document 1, Non-Patent Document 1) However, since the above blocking agents are proteins derived from living organisms, there is a potential problem that performance is prone to variation.

[0003] Generally, the substrates used in immunodiagnosis are flat substrates such as glass or polystyrene, or particles such as latex or gold colloid. The two have different surface roughness, and when a flat substrate is used, there is a problem that the effect of suppressing nonspecific reactions is difficult to exert unless the affinity between the substrate and the blocking agent is sufficient.

[0004] In immunocytochemical staining, cells are cultured on a cover glass, and in immunohistochemical staining, tissue sections are attached to a slide glass to prepare specimens. In order to improve the adhesion of cells and tissues to these cover glasses and slide glasses, coating agents such as poly-L-lysine, MAS Coat (Matsunami Glass Industry Co., Ltd.), and 3-aminopropyltriethoxysilane (APS) are applied to these cover glasses and slide glasses, and the amino groups of the coating agent impart a positive charge to the glass surface. In the immunostaining method, during the color development process, the cover glasses and slide glasses coated with the above-mentioned coating agents may be colored by the color-developing dye in the reagent (Patent Document 2). This phenomenon occurs because the blocking process is not performed appropriately, and as a result, it becomes difficult to determine whether the specimen is colored, which is problematic.

[0005] In order to solve the above problems, blocking agents of non-biological origin and blocking agents that prevent slide staining have been investigated. For example, Patent Document 2 discloses that BSA is succinylated to prevent slide staining. Patent Document 3 and Non-Patent Document 2 disclose a non-biological blocking agent using a copolymer having a phosphorylcholine group in the side chain. Patent Documents 4, 5, and 6 also disclose a surfactant called Tween (registered trademark) 20, hydroxyalkyl cellulose, polyvinyl alcohol, copolymers of (meth)acryloylmorpholine and other monomers, and the like. Furthermore, Patent Document 7 discloses a non-specific adsorption inhibitor that contains a resin having a zwitterionic structure as an active ingredient. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2016 / 052690 [Patent Document 2] International Publication No. 2019 / 130560 [Patent Document 3] Japanese Patent Application Publication No. 07-083923 [Patent Document 4] JP 2004-219111 A [Patent Document 5] Japanese Patent Application Publication No. 04-019561 [Patent Document 6] JP 2008-209114 A [Patent Document 7] JP 2019-99705 A [Non-patent literature]

[0007] [Non-Patent Document 1] "Watanabe and Nakane, Enzyme Immunosorbent Assay" published by Gakusai Kikaku Co., Ltd., 2002 [Non-Patent Document 2] Polymer Journal Vol. 35 No. 7 423(1978) Summary of the Invention [Problem to be solved by the invention]

[0008] However, many of the conventional blocking agents are not sufficient in preventing non-specific adsorption. In addition, proteins derived from living organisms, particularly those disclosed in Patent Document 2, have a potential problem of being prone to variation in performance. Furthermore, when applied to antigen-antibody reactions carried out on a planar substrate, conventional blocking agents are insufficient in preventing non-specific adsorption, suppressing slide coloring, stabilizing proteins, and / or enhancing color development in immunostaining.

[0009] In view of the above circumstances, an object of the present invention is to provide a blocking agent, a kit, and a blocking method that have excellent non-specific adsorption prevention ability, slide coloration suppression ability, protein stabilization ability, and color development sensitization effect in immunostaining methods. [Means for solving the problem]

[0010] That is, the present invention relates to the following [1] to

[14] .

[0011] [1] A blocking agent for preventing nonspecific adsorption in an antigen-antibody reaction carried out on a planar substrate, Contains a water-soluble vinyl polymer (A), The water-soluble vinyl polymer (A) contains the following structural unit (a), structural unit (b), and structural unit (c), The blocking agent has a content of the structural unit (c) of 5 to 35% by mass in 100% by mass of the water-soluble vinyl polymer (A). Structural unit (a): A structural unit having a zwitterionic structure Structural unit (b): A structural unit derived from a monomer (b') having a partition coefficient LogP of 0.8 to 10. Structural unit (c): a structural unit derived from a monomer (c') having a pKa in water of -3 to 5 (excluding those having a zwitterionic structure)

[0012] [2] The blocking agent according to [1], wherein the water-soluble vinyl polymer (A) has an acid value of 20 to 250 mgKOH / g.

[0013] [3] The blocking agent according to [1] or [2], wherein the surface zeta potential of a slide glass having a surface zeta potential of +10 mV when covered with a 1 w / v % aqueous solution of the water-soluble vinyl polymer (A) is -100 to 0 mV.

[0014] [4] The blocking agent according to any one of [1] to [3], wherein the water-soluble vinyl polymer (A) has a pH of 3 to 7 in a 1 w / v % aqueous solution.

[0015] [5] The blocking agent according to any one of [1] to [4], wherein the structural unit (c) has one or more functional groups selected from the group consisting of a carboxy group, a sulfo group, and a phosphate group.

[0016] [6] The blocking agent according to any one of [1] to [5], wherein the structural unit (a) has one or more zwitterionic structures selected from the group consisting of phosphorylcholine, sulfobetaine, carbobetaine, and amine oxide.

[0017] [7] The blocking agent according to [6], wherein the structural unit (a) has one or more zwitterionic structures selected from the group consisting of the following formulae (1) to (7): General formula (1) [ka] General formula (2) [ka] General formula (3) [ka] General formula (4) [ka] General formula (5) [ka] General formula (6) [ka] General formula (7) [ka] [In general formulas (1) to (7), X is an oxygen atom or NH; Y is COO - or SO3 - , R2, R 16 , R 27 each independently represents an alkylene group having 1 to 6 carbon atoms, R3, R4, R 17 , R 18 , R 29 , R 30 , R 31 each independently represents an alkyl group having 1 to 4 carbon atoms; R5, R 28 each independently represents an alkylene group having 1 to 4 carbon atoms, R7, R 20 each independently represents a hydrogen atom or a methyl group, R8 is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms; R9~R 13 Four of them, R 21 ~R 25 four of the groups are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R 14 represents an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms, R9~R 13 One of them, R 21 ~R 25 one of the groups represents a bonding site with the main chain of the vinyl polymer, * indicates the bonding position with the main chain of the vinyl polymer.]

[0018] [8] The blocking agent according to any one of [1] to [7], wherein the content of the structural unit (a) is 35 to 85 mass % in 100 mass % of the water-soluble vinyl polymer (A).

[0019] [9] The blocking agent according to any one of [1] to [8], wherein the content of the structural unit (b) is 10 to 50 mass % in 100 mass % of the water-soluble vinyl polymer (A).

[0020]

[10] The blocking agent according to any one of [1] to [9], wherein the water-soluble vinyl polymer (A) has a mass average molecular weight of 2,000 to 1,000,000.

[0021]

[11] The blocking agent according to any one of [1] to

[10] , further comprising water, wherein the concentration of the water-soluble vinyl polymer (A) is 0.01 to 30.0 w / v %.

[0022]

[12] A kit comprising the blocking agent according to any one of [1] to

[11] and a planar substrate.

[0023]

[13] The kit according to

[12] , wherein the surface zeta potential of the planar substrate is 0 to +50 mV before the blocking treatment.

[0024]

[14] A blocking method comprising the step of subjecting a flat substrate to a blocking treatment with the blocking agent according to any one of [1] to

[11] . Effect of the Invention

[0025] The present invention makes it possible to provide a blocking agent, a kit, and a blocking method that have excellent abilities to prevent non-specific adsorption, inhibit slide staining, stabilize proteins, and enhance color development in immunostaining. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] <Blocking agent> The blocking agent of the present invention is a blocking agent for preventing non-specific adsorption in an antigen-antibody reaction carried out on a planar substrate. In the present invention, blocking refers to preventing nonspecific adsorption caused by factors other than the antigen-antibody reaction between a target substance in a specimen and an antigen or antibody against the target substance in in vitro diagnosis, preventing the adsorption of an antibody, antigen, or color-developing dye to a planar substrate, and preventing nonspecific adsorption caused by factors other than these reactions. Note that if a target substance in a specimen is adsorbed to a substrate without the intervention of an antigen or antibody against the target substance, abnormalities in the measured value may occur, and this also includes preventing such abnormalities. Examples of the in vitro diagnosis include diagnosis by immunostaining, ELISA, Western blotting (WB), etc.

[0027] The specimens typically include cell tissues, various biological liquid samples such as serum, plasma, urine, saliva, etc., pulverized specimens of feces or food, etc. As the measurement sample, a specimen dilution solution prepared by diluting the specimen with a pH buffer, protein, amino acid, etc. may be used.

[0028] [pH] The pH of the blocking agent of the present invention is preferably 3 to 7, more preferably 3.5 to 7, more preferably 4 to 7, and even more preferably 4.5 to 7. When the pH of the blocking agent is within the above range, excess outflow of the blocking agent in the washing step can be prevented, and the blocking effect can be exerted.

[0029] [Surface zeta potential] When the blocking agent of the present invention is applied to a slide glass substrate having a surface zeta potential of +10 mV, the surface zeta potential of the substrate after coating is preferably −100 to 0 mV, more preferably −85 to 0 mV, and even more preferably −70 to 0 mV. When the surface zeta potential is within the above range, the water-soluble vinyl polymer (A) in the blocking agent adheres closely to the substrate, and adsorption of the color-developing dye to the substrate and nonspecific adsorption due to antigen-antibody reaction can be suppressed.

[0030] The above surface zeta potential can be measured by immersing a slide glass in a blocking agent at room temperature for 30 minutes, washing it three times with pure water, and then using an electrophoretic light scattering method. Specifically, the slide glass after the above immersion was cut to the size of the sample holder (approximately 10 mm x 35 mm) of the flat plate zeta potential measurement cell, and used as the measurement sample. The standard particle aqueous solution was placed in the flat plate zeta potential measurement cell, and the sample holder with the sample set was immersed in it. An electric field was applied to the standard particles, and the zeta potential of the sample surface was obtained from the mobility of the standard particles, which changes depending on the condition of the sample surface.

[0031] <Water-soluble vinyl polymer (A)> The blocking agent of the present invention contains a water-soluble vinyl polymer (A). By coating an antigen, a planar substrate, or a planar carrier on which an antibody is immobilized against a target substance in a specimen with the water-soluble vinyl polymer (A), the ability to suppress non-specific color development in the specimen and the ability to prevent adsorption of a dye to the substrate can be stably exhibited. In the present invention, among planar substrates, a support for immobilizing an antibody or an antigen is referred to as a planar carrier. In the present invention, water-solubility refers to the state in which, after 1 g of resin is placed in 100 mL of ion-exchanged water at 25° C., stirred, and left to stand at 25° C. for 24 hours, the resin is completely soluble in water without separation or precipitation.

[0032] The water-soluble vinyl polymer (A) contains the following structural units (a), (b), and (c). Structural unit (a): A structural unit having a zwitterionic structure Structural unit (b): A structural unit derived from a monomer (b') having a partition coefficient LogP of 0.8 to 10. Structural unit (c): a structural unit derived from a monomer (c') having a pKa in water of -3 to 5 (excluding those having a zwitterionic structure)

[0033] [Structural unit (a)] The structural unit (a) is a structural unit having a zwitterionic structure, is preferably a structural unit derived from a monomer (a') having a zwitterionic structure, and preferably has a repeating structure. A zwitterionic structure is a structure that has positive and negative charges in the same molecule, and the positively charged atom does not have a dissociable hydrogen atom bonded to it. A structure that has a positive charge and a negative charge in non-adjacent positions in the same molecule is called a betaine structure. When the water-soluble vinyl polymer (A) has this zwitterionic structure in the molecule, the adsorption of the blocking agent to proteins is controlled, and excellent blocking performance can be achieved.

[0034] The content of the structural unit (a) is preferably 35 to 85% by mass, more preferably 35 to 75% by mass, further preferably 45 to 65% by mass, and particularly preferably 50 to 60% by mass, based on 100% by mass of the water-soluble vinyl polymer (A). When the content of the structural unit (a) is within the above range, water solubility and nonspecific adsorption of proteins can be suppressed, and excellent blocking performance can be exhibited.

[0035] The zwitterionic structure of the structural unit (a) is preferably one or more selected from the group consisting of phosphorylcholine, sulfobetaine, carbobetaine, and amine oxide, more preferably one or more selected from the group consisting of sulfobetaine, carbobetaine, and amine oxide, and particularly preferably an amine oxide.

[0036] Furthermore, the zwitterionic structure possessed by the structural unit (a) is preferably one or more selected from the group consisting of the following general formulae (1) to (7).

[0037] General formula (1) [ka]

[0038] General formula (2) [ka]

[0039] General formula (3) [ka]

[0040] General formula (4) [ka]

[0041] General formula (5) [ka]

[0042] General formula (6) [ka]

[0043] General formula (7) [ka]

[0044] [In general formulas (1) to (7), X is an oxygen atom or NH; Y is COO - or SO3 -, R2, R 16 , R 27 each independently represents an alkylene group having 1 to 6 carbon atoms, R3, R4, R 17 , R 18 , R 29 , R 30 , R 31 each independently represents an alkyl group having 1 to 4 carbon atoms; R5, R 28 each independently represents an alkylene group having 1 to 4 carbon atoms, R7, R 20 each independently represents a hydrogen atom or a methyl group, R8 is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms; R9~R 13 Four of them, R 21 ~R 25 four of the groups are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R 14 represents an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms, R9~R 13 One of them, R 21 ~R 25 one of the groups represents a bonding site with the main chain of the vinyl polymer, * indicates the bonding position with the main chain of the vinyl polymer.]

[0045] Methods for obtaining a water-soluble vinyl polymer (A) having a zwitterionic structure, which is the structural unit (a), include [1] a method using a monomer (a') having a zwitterionic structure in polymerization, and [2] a method using a monomer (a'') which does not have a zwitterionic structure and has a reactive site with a betaining agent (D) or an oxidizing agent (E) described later in polymerization, and then introducing the zwitterionic structure later. Specifically, the following methods are preferred. [1] At least one of the monomers (a'1) to (a'7) represented by the following general formulas (8) to (14) is copolymerized with other monomers as required. or, [2] At least one of the monomers (a''1) to (a''3) represented by the following general formulas (15) to (17) is copolymerized with other monomers as necessary, and the portions derived from the monomers (a''1) to (a''3) in the obtained copolymer (at least a portion of the nitrogens represented by ** in the following general formulas (15) to (17)) are reacted with a betaining agent (D) or an oxidizing agent (E) described below. The obtained reaction product has a zwitterionic structure, similar to the copolymers using the monomers (a'1) to (a'7). Monomers having a zwitterionic structure are highly water-soluble and have limitations on copolymerization with other monomers. Therefore, a more preferable method is to use a monomer (a'') that does not have a zwitterionic structure and has a reactive site with a betaining agent (D) or an oxidizing agent (E) described later for polymerization, and then introduce the zwitterionic structure later.

[0046] [Monomers (a') and (a'') having zwitterionic structures] The monomer (a') having a zwitterionic structure is preferably one or more selected from the group consisting of the following monomers (a'1) to (a'7), and (a'') is preferably one or more selected from the group consisting of (a''1) to (a''3).

[0047] Monomer (a'1) General formula (8) [ka]

[0048] Monomer (a'2) General formula (9) [ka]

[0049] Monomer (a'3) General formula (10) [ka]

[0050] Monomer (a'4) General formula (11) [ka]

[0051] Monomer (a'5) General formula (12) [ka]

[0052] Monomer (a'6) General formula (13) [ka]

[0053] Monomer (a'7) General formula (14) [ka]

[0054] Monomer (a''1) General formula (15) [ka]

[0055] Monomer (a''2) General formula (16) [ka]

[0056] Monomer (a''3) General formula (17) [ka]

[0057] [In general formulas (8) to (17), X is an oxygen atom or NH; Y is COO- or SO3 - , R1R6, R7, R 15 , R 19 , R 20 , R 26 is a hydrogen atom or a methyl group R2, R 16 , R 27 each independently represents an alkylene group having 1 to 6 carbon atoms, R3, R4, R 17 , R 18 , R 29 , R 30 , R 31 each independently represents an alkyl group having 1 to 4 carbon atoms; R5, R 28 is an alkylene group having 1 to 4 carbon atoms, R8 is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms; R9~R 13、 R 21 ~R 25 four of the groups are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R 14 represents an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms, R9~R 13、 R 21 ~R 25 One of them is CH2=CH ** indicates a reaction site with a betaine agent (D) or an oxidizing agent (E).]

[0058] As represented by the general formula (8), the monomer (a'1) has one ethylenically unsaturated group and a betaine structure in one molecule. Examples of such monomers include N-(meth)acryloyloxymethyl-N,N-dimethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxyethyl-N,N-dimethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxypropyl-N,N-dimethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxybutyl-N,N-dimethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxymethyl-N,N-diethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxyethyl-N,N-diethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxypropyl-N,N-diethylammonium methyl-α-carboxybetaine, N-(meth)acryloyloxyalkyl-N,N-dialkylammonium alkyl-α-carboxybetaine, N-(meth)acryloyloxybutyl-N,N-diethylammonium methyl-α-carboxybetaine, etc.; N-(meth)acrylamidoalkyl-N,N-dialkylammonium alkyl-α-carboxybetaine, N-(meth)acrylamidopropyl-N,N-dimethylammonium methyl-α-carboxybetaine, N-(meth)acrylamidopropyl-N,N-diethylammonium methyl-α-carboxybetaine, etc.; N-(meth)acrylamidoalkyl-N,N-dialkylammonium alkyl-α-carboxybetaine, N-(meth)acrylamidopropyl-N,N-dimethylammonium methyl-α-carboxybetaine, N-(meth)acrylamidopropyl-N,N-diethylammonium methyl-α-carboxybetaine, etc.;N-(meth)acryloyloxymethyl-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxymethyl-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxymethyl-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxymethyl-N,N-dimethylammonium butyl-α-sulfobetaine, N-(meth)acryloyloxyethyl-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxyethyl-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxyethyl-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxyethyl-N,N-dimethylammonium butyl-α-sulfobetaine, N-(meth)acryloyloxypropyl-N,N-dimethyl N-(meth)acryloyloxyalkyl-N,N-dimethylammonium alkyl-α-sulfobetaine, N-(meth)acryloyloxypropyl-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxypropyl-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxypropyl-N,N-dimethylammonium butyl-α-sulfobetaine, N-(meth)acryloyloxybutyl-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxybutyl-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxybutyl-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxybutyl-N,N-dimethylammonium butyl-α-sulfobetaine, etc. N-(meth)acryloyloxyalkyl-N,N-dimethylammonium alkyl-α-sulfobetaine;N-(meth)acryloyloxymethoxymethoxy-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxymethoxymethoxy-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxymethoxymethoxy-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxymethoxymethoxy-N,N-dimethylammonium butyl-α-sulfobetaine, N-(meth)acryloyloxyethoxy N-(meth)acryloyloxyethoxyethoxy-N,N-dimethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxyethoxyethoxy-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxyethoxyethoxy-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxyethoxyethoxy-N,N-dimethylammonium butyl-α-sulfobetaine, N-(meth)acryloyloxypropoxypropoxy-N,N-dimethyl ethylammonium methyl-α-sulfobetaine, N-(meth)acryloyloxypropoxypropoxy-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxypropoxypropoxy-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxypropoxypropoxy-N,N-dimethylammonium butyl-α-sulfobetaine, N-(meth)acryloyloxybutoxybutoxy-N,N-dimethylammonium methyl -α-sulfobetaine, N-(meth)acryloyloxybutoxybutoxy-N,N-dimethylammonium ethyl-α-sulfobetaine, N-(meth)acryloyloxybutoxybutoxy-N,N-dimethylammonium propyl-α-sulfobetaine, N-(meth)acryloyloxybutoxybutoxy-N,N-dimethylammonium butyl-α-sulfobetaine, etc. N-(meth)acryloyloxyalkoxyanerkoxy-N,N-dimethylammonium alkyl-α-sulfobetaine;Examples of the N-(meth)acrylamidoalkyl-N,N-dialkylammonium alkyl-α-sulfobetaine include N-(meth)acrylamidopropyl-N,N-dimethylammonium propyl-α-sulfobetaine and N-(meth)acrylamidopropyl-N,N-dimethylammonium butyl-α-sulfobetaine. In the present invention, the term "(meth)acryl" refers to methacryl or acrylic.

[0059] As shown in the general formula (9), the monomer (a'2) also has one ethylenically unsaturated group and a betaine structure in one molecule. Examples of such monomers include 1-vinyl-2-alkyl-3-(4-sulfoalkyl)imidazolium inner salts such as 1-vinyl-3-(3-sulfopropyl)imidazolium inner salt, 1-vinyl-3-(3-sulfobutyl)imidazolium inner salt, 1-vinyl-2-methyl-3-(3-sulfopropyl)imidazolium inner salt, and 1-vinyl-2-methyl-3-(4-sulfobutyl)imidazolium inner salt.

[0060] As shown in the general formula (10), the monomer (a'3) also has one ethylenically unsaturated group and a betaine structure in one molecule. Examples of such monomers include 1-vinyl-2-alkyl-3-(4-sulfoalkyl)imidazolium inner salts such as 1-vinyl-3-(3-sulfopropyl) inner salt, 1-vinyl-3-(3-sulfobutyl)pyridinium inner salt, 1-vinyl-2-methyl-3-(3-sulfopropyl)pyridinium inner salt, and 1-vinyl-2-methyl-3-(4-sulfobutyl)pyridinium inner salt.

[0061] As represented by the general formula (11), the monomer (a'4) also has one ethylenically unsaturated group and a betaine structure in one molecule. Examples of such monomers include 2-((meth)acryloyloxy)ethyl-2'-(trimethylammonio)ethyl phosphate, 3-((meth)acryloyloxy)propyl-2'-(trimethylammonio)ethyl phosphate, 4-((meth)acryloyloxy)butyl-2'-(trimethylammonio)ethyl phosphate, 5-((meth)acryloyloxy)pentyl-2'-(trimethylammonio)ethyl phosphate, 6-((meth)acryloyloxy)hexyl-2'-(trimethylammonio)ethyl phosphate, 2-((meth)acryloyloxy)ethyl-2'-(triethylammonio)ethyl phosphate, 2-((meth)acryloyloxy)ethyl-2'-(tripropylammonio)ethyl phosphate, 2-((meth)acryloyloxy)ethyl-2'-(tributylammonio)ethyl phosphate phosphate, 2-((meth)acryloyloxy)ethyl-2'-(tricyclohexylammonio)ethyl phosphate, 2-((meth)acryloyloxy)ethyl-2'-(triphenylammonio)ethyl phosphate, 2-((meth)acryloyloxy)ethyl-2'-(trimethanolammonio)ethyl phosphate, 2-((meth)acryloyloxy)propyl-2'-(trimethylammonio)ethyl phosphate phosphate, 2-((meth)acryloyloxy)butyl-2'-(trimethylammonio)ethyl phosphate, 2-((meth)acryloyloxy)pentyl-2'-(trimethylammonio)ethyl phosphate, 2-((meth)acryloyloxy)hexyl-2'-(trimethylammonio)ethyl phosphate, 2-(vinyloxy)ethyl-2'-(trimethylammonio)ethyl phosphate, 2-(allyloyloxy)ethyl 2-(p-vinylbenzyloxy)ethyl-2'-(trimethylammonio)ethyl phosphate, 2-(p-vinylbenzoyloxy)ethyl-2'-(trimethylammonio)ethyl phosphate, 2-(styryloxy)ethyl-2'-(trimethylammonio)ethyl phosphate, 2-(p-vinylbenzyloxy)ethyl-2'-(trimethylammonio)ethyl phosphate, 2-Examples of the hydroxyethyl-(2-trimethylammonioethylphosphorylethyl)malate include (vinyloxycarbonyl)ethyl-2'-(trimethylammonio)ethyl phosphate, 2-(allyloxycarbonyl)ethyl-2'-(trimethylammonio)ethyl phosphate, 2-((meth)acryloylamino)ethyl-2'-(trimethylammonio)ethyl phosphate, 2-(vinylcarbonylamino)ethyl-2'-(trimethylammonio)ethyl phosphate, ethyl-(2-trimethylammonioethylphosphorylethyl)fumarate, butyl-(2-trimethylammonioethylphosphorylethyl)fumarate, hydroxyethyl-(2-trimethylammonioethylphosphorylethyl)fumarate, ethyl-(2-trimethylammonioethylphosphorylethyl)malate, butyl-(2-trimethylammonioethylphosphorylethyl)malate, and hydroxyethyl-(2-trimethylammonioethylphosphorylethyl)malate.

[0062] As shown in general formula (12), the monomer (a'5) has one ethylenically unsaturated group and a betaine structure in one molecule. Examples of such monomers include N-(meth)acryloyloxymethyl-N,N-dimethylamine-N-oxide, N-(meth)acryloyloxyethyl-N,N-dimethylamine-N-oxide, N-(meth)acryloyloxypropyl-N,N-dimethylamine-N-oxide, N-(meth)acryloyloxybutyl-N,N-dimethylamine-N-oxide, N-(meth)acryloyloxyalkyl-N,N-dialkylamine-N-oxides such as N-(meth)acryloyloxymethyl-N,N-diethylamine-N-oxide, N-(meth)acryloyloxyethyl-N,N-diethylamine-N-oxide, N-(meth)acryloyloxypropyl-N,N-diethylamine-N-oxide, and N-(meth)acryloyloxybutyl-N,N-diethylamine-N-oxide; N-(meth)acrylamidopropyl-N,N-dimethylamine-N-oxide; Examples of the compound include N-(meth)acrylamidoalkyl-N,N-dialkylamine-N-oxides such as N-(meth)acrylamidopropyl-N,N-diethylamine-N-oxide; and N-(meth)acryloyloxyalkoxyanelkoxy-N,N-dimethylamine-N-oxides such as N-(meth)acryloyloxymethoxymethoxy-N,N-dimethylamine-N-oxide, N-(meth)acryloyloxyethoxyethoxy-N,N-dimethylamine-N-oxide, N-(meth)acryloyloxypropoxypropoxy-N,N-dimethylamine-N-oxide, and N-(meth)acryloyloxybutoxybutoxy-N,N-dimethylamine-N-oxide.

[0063] As represented by the general formula (13), the monomer (a'6) has one ethylenically unsaturated group and a betaine structure in one molecule. Examples of such monomers include amine oxides such as 1-vinylimidazole, 2-methyl-1-vinylimidazole, 4-methyl-1-vinylimidazole, 5-methyl-1-vinylimidazole, 2-lauryl-1-vinylimidazole, and 4-(t-butyl)-1-vinylimidazole.

[0064] As shown in the general formula (14), the monomer (a'7) has one ethylenically unsaturated group and a betaine structure in one molecule. Examples of such monomers include amine oxides such as 2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, 2-methyl-3-vinylpyridine, 2-methyl-4-vinylpyridine, 3-methyl-4-vinylpyridine, 2-methyl-5-vinylpyridine, 3-methyl-5-vinylpyridine, 4-methyl-5-vinylpyridine, 2-lauryl-4-vinylpyridine, 2-lauryl-5-vinylpyridine, 2-(t-butyl)-4-vinylpyridine, and 2-(t-butyl)-5-vinylpyridine.

[0065] Examples of the monomer (a''1) include N-(meth)acryloyloxyalkyl-N,N-dialkylamines such as N-(meth)acryloyloxymethyl-N,N-dimethylamine, N-(meth)acryloyloxyethyl-N,N-dimethylamine, N-(meth)acryloyloxypropyl-N,N-dimethylamine, N-(meth)acryloyloxybutyl-N,N-dimethylamine, N-(meth)acryloyloxymethyl-N,N-diethylamine, N-(meth)acryloyloxyethyl-N,N-diethylamine, N-(meth)acryloyloxypropyl-N,N-diethylamine, and N-(meth)acryloyloxybutyl-N,N-diethylamine; N-(meth)acrylamidopropyl-N,N-dimethylamine; Examples of such N-(meth)acrylamidoalkyl-N,N-dialkylamines include N-(meth)acrylamidopropyl-N,N-diethylamine and the like; and N-(meth)acryloyloxyalkoxyanelkoxy-N,N-dimethylamines such as N-(meth)acryloyloxymethoxymethoxy-N,N-dimethylamine, N-(meth)acryloyloxyethoxyethoxy-N,N-dimethylamine, N-(meth)acryloyloxypropoxypropoxy-N,N-dimethylamine and N-(meth)acryloyloxybutoxybutoxy-N,N-dimethylamine.

[0066] Examples of the monomer (a''2) include 1-vinylimidazole, 2-methyl-1-vinylimidazole, 4-methyl-1-vinylimidazole, 5-methyl-1-vinylimidazole, 2-lauryl-1-vinylimidazole, and 4-(t-butyl)-1-vinylimidazole.

[0067] Examples of the monomer (a''3) include 2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine, 2-methyl-3-vinylpyridine, 2-methyl-4-vinylpyridine, 3-methyl-4-vinylpyridine, 2-methyl-5-vinylpyridine, 3-methyl-5-vinylpyridine, 4-methyl-5-vinylpyridine, 2-lauryl-4-vinylpyridine, 2-lauryl-5-vinylpyridine, 2-(t-butyl)-4-vinylpyridine, and 2-(t-butyl)-5-vinylpyridine.

[0068] <Betaine agent (D)> The betaining agent (D) is a group of compounds used for sulfobetaining or carbobetaining the nitrogen represented by ** in the monomers (a''1) to (a''3) represented by the general formulas (15) to (17) after polymerization. The betaining agent (D) is preferably selected from the group consisting of cyclic sulfonate esters (D1), ω-halogenated alkylsulfonate metal salts (D2), cyclic carboxylate esters (D3) and ω-halogenated alkylcarboxylate metal salts (D4).

[0069] Examples of such cyclic sulfonic acid esters (D1) include 1,2-ethane sultone, 1,3-propane sultone, and 1,4-butane sultone.

[0070] Examples of such ω-halogenated alkylsulfonic acid metal salts (D2) include sodium 2-chloroethanesulfonate, sodium 2-bromoethanesulfonate, sodium 3-chloropropanesulfonate, sodium 3-bromopropanesulfonate, sodium 4-chlorobutanesulfonate, and sodium 4-bromobutanesulfonate.

[0071] Examples of such cyclic carboxylates (D3) include β-propiolactone, γ-butyrolactone, and δ-valerolactone.

[0072] Examples of such ω-halogenated alkyl carboxylate metal salts (D4) include sodium 2-chloroacetate, sodium 2-bromoacetate, sodium 3-chloropropionate, sodium 3-bromopropionate, sodium 4-chlorobutyrate, sodium 4-bromobutyrate, sodium 5-chloropentanoate, and sodium 5-bromopentanoate.

[0073] [Oxidizing agent (E)] The oxidizing agent (E) is a group of compounds used to oxidize the nitrogen represented by ** in the monomers (a''1) to (a''3) represented by the general formulas (15) to (17) after polymerization. As the oxidizing agent (E), an oxidizing agent such as peroxide or ozone is used. Examples of peroxides include hydrogen peroxide, ammonium persulfate, sodium persulfate, peracetic acid, metachloroperbenzoic acid, benzoyl peroxide, and t-butyl hydroperoxide. Of these, hydrogen peroxide is preferred and is usually used in the form of an aqueous solution.

[0074] [Structural unit (b)] The water-soluble vinyl polymer (A) contains a structural unit derived from a monomer (b') having a partition coefficient LogP of 0.8 to 10. The introduction of the structural unit based on the monomer (b') enhances the adhesion to the substrate and the sample, and allows the blocking agent to remain on the substrate even after a washing step.

[0075] The content of the structural unit (b) is preferably 10 to 50% by mass, more preferably 15 to 50% by mass, and even more preferably 25 to 40% by mass, based on 100% by mass of the water-soluble vinyl polymer (A). When the content of the structural unit (b) is within the above range, the water solubility can be appropriately controlled. In addition, the adhesion between the substrate and the specimen is increased by hydrophobic interaction, and the blocking agent can be retained on the substrate even after a washing process.

[0076] [Monomer (b')] Examples of the monomer (b') having a distribution coefficient LogP of 0.8 to 10 include methyl methacrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-butyl (meth)acrylate, pentyl (meth)acrylate, heptyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, and dodecyl (meth)acrylate. Examples of the acrylate (meth)acrylate include alkyl ester (meth)acrylates such as acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, isostearyl (meth)acrylate, behenyl (meth)acrylate, cyclohexyl (meth)acrylate, and isobornyl (meth)acrylate; aromatic ester (meth)acrylates such as phenyl (meth)acrylate, benzyl (meth)acrylate, and phenoxyethyl (meth)acrylate; and aromatic vinyl monomers such as styrene, α-methylstyrene, 2-methylstyrene, chlorostyrene, allylbenzene, and ethynylbenzene. These may be used alone or in combination of two or more kinds.

[0077] The partition coefficient LogP is a numerical value that indicates the properties of a chemical substance, and is a constant value that does not depend on the amount added. It is the common logarithm of the concentration of each phase when the substance is in equilibrium in a mixture of water and 1-octanol, where the aqueous phase and the octanol phase are in contact with each other. When LogP is large, the substance tends to be relatively more hydrophobic, and when LogP is small, the substance tends to be relatively more hydrophilic.

[0078] LogP can be measured by the flask shaking method described in JIS Japanese Industrial Standards Z7260-107 (2000). LogP can also be estimated by computational chemistry methods or empirical methods instead of actual measurements. Known methods and software can be used to calculate LogP, but in the present invention, LogP is calculated using a program incorporated in CambridgeSoft's system: ChemdrawPro11.0.

[0079] Structural Units (c) The water-soluble vinyl polymer (A) contains a structural unit (excluding those having a zwitterionic structure) derived from a monomer (c') having a pKa in water of -3 to 5. By introducing a structural unit based on the monomer (c'), the adhesion to the substrate is controlled, and the blocking effect on the flat substrate can be maximized.

[0080] The content of the structural unit (c) is preferably 5 to 35% by mass, more preferably 10 to 35% by mass, in 100% by mass of the water-soluble vinyl polymer (A). When the content is within the above range, adhesion to the substrate and cells is enhanced by electrostatic interaction, and the blocking effect can be maximized.

[0081] [Monomer (c')] Preferred examples of the monomer (c') having a pka in water of -3 to 5 include monomers having one or more functional groups selected from the group consisting of a carboxy group, a sulfo group, and a phosphate group, more preferably monomers having a carboxy group or a sulfo group, and particularly preferably monomers having a carboxy group. More specifically, for example, acrylic acid, methacrylic acid, 2-(trifluoromethyl)acrylic acid, mono(2-acryloyloxyethyl) succinate, monohydroxyethyl phthalate acrylate, ω-carboxy-polycaprolactone monoacrylate, mono-2-(methacryloyloxy)ethyl phthalate, 6-acrylamidohexanoic acid, 4-carboxystyrene, maleic acid, fumaric acid, itaconic acid, citraconic acid, alkenyl monoester, isophthalic acid β-(meth)acryloxyethyl monoester, terephthalic acid β-( Examples of the monomer include monomers having a carboxy group, such as β-(meth)acryloxyethyl monoester of succinic acid, acrylic acid, methacrylic acid, crotonic acid, and cinnamic acid; monomers having a sulfo group, such as vinyl sulfonic acid, sodium p-styrenesulfonate, and Nt-butylacrylamidosulfonic acid; and monomers having a phosphoric acid group, such as mono(2-hydroxyethyl methacrylate) phosphate, 2-acryloyloxyethyl acid phosphate, and 2-methacryloyloxyethyl acid phosphate.

[0082] The acid dissociation constant pKa is a value that quantitatively represents the acidity of a chemical substance, and the smaller the pKa, the stronger the acidity. In the present invention, pKa is calculated using a program incorporated in CambridgeSoft's system: ChemdrawPro11.0.

[0083] [Acid value] The acid value of the water-soluble vinyl polymer (A) is preferably 20 to 250 mgKOH / g, more preferably 25 to 220 mgKOH / g, more preferably 30 to 200 mgKOH / g, and even more preferably 50 to 150 mgKOH / g. When the acid value of the water-soluble vinyl polymer (A) is within the above range, adhesion to a substrate or a specimen is increased by electrostatic interaction, and the blocking effect can be maximized.

[0084] [pH] The pH of a 1 w / v % aqueous solution of the water-soluble vinyl polymer (A) is preferably 3 to 7, more preferably 3.5 to 7, more preferably 4 to 7, and even more preferably 4.5 to 7. When the pH of a 1 w / v % aqueous solution of the water-soluble vinyl polymer (A) is within the above range, excess outflow of the blocking agent in the washing step can be prevented, and the blocking effect can be exerted.

[0085] [Mass average molecular weight] The mass average molecular weight of the water-soluble vinyl polymer (A) is preferably 2,000 to 1,000,000, more preferably 5,000 to 500,000, and even more preferably 1,0000 to 150,000. When the mass average molecular weight of the water-soluble vinyl polymer (A) is within the above range, the adsorption of the blocking agent to the substrate or the sample can be appropriately controlled. In addition, the appropriate viscosity allows the handling properties to be maintained.

[0086] [Surface zeta potential] When the water-soluble vinyl polymer (A) is used in a 1 w / v % aqueous solution to coat a slide glass substrate having a surface zeta potential of +10 mV, the surface zeta potential of the substrate after coating is preferably −10 to 0 mV, more preferably −85 to 0 mV, and even more preferably −70 to 0 mV. When the surface zeta potential is within the above range, the water-soluble vinyl polymer (A) adheres closely to the substrate, and adsorption of the color-developing dye to the substrate and nonspecific adsorption due to antigen-antibody reaction can be suppressed.

[0087] The surface zeta potential of the above slide glass was measured by immersing the slide glass in a 1 w / v % aqueous solution of the water-soluble vinyl polymer (A) at room temperature for 30 minutes, washing it three times with pure water, and then using an ELSZ-2000Z manufactured by Otsuka Electronics Co., Ltd., by electrophoretic light scattering method. Specifically, the slide glass after the above immersion was cut to the size of the sample holder (approximately 10 mm x 35 mm) of the flat plate zeta potential measurement cell, and used as the measurement sample. The standard particle aqueous solution was placed in the flat plate zeta potential measurement cell, and the sample holder with the sample set was immersed in it. An electric field was applied to the standard particles, and the zeta potential of the sample surface was obtained from the mobility of the standard particles, which changes depending on the condition of the sample surface.

[0088] [Aqueous medium] The blocking agent of the present invention preferably further contains an aqueous medium in addition to the water-soluble vinyl polymer (A). When the blocking agent contains an aqueous medium, the concentration of the water-soluble vinyl polymer (A) is preferably 0.01 to 30.0 w / v%, more preferably 0.01 to 15 w / v%, and even more preferably 0.1 to 5 w / v%. By being in the above range, it is possible to maintain the handleability due to the appropriate viscosity and to exert the effect of suppressing non-specific adsorption.

[0089] The aqueous medium preferably contains water, and may be a buffer solution such as PBS or PBS-T.

[0090] <Kit> The kit of the present invention comprises the above-mentioned blocking agent and a planar substrate.

[0091] [Plane base material] The flat substrate refers to a member that can be placed horizontally, and does not exclude substrates with slight curvature, but preferably has no visible curvature. The radius of curvature of the flat substrate is more preferably 100 mm or more, even more preferably 1000 mm or more, and particularly preferably 10000 mm or more. The form of the flat substrate may be an ELISA plate, an immunochromatography membrane, a Western blotting membrane substrate, an immunostaining slide glass, a cover glass, etc.

[0092] The material of the flat substrate is not particularly limited, but examples thereof include nitrocellulose, nylon, polyvinylidene fluoride (PVDF), polyolefin, polycycloolefin, polyester, inorganic glass, carbon, silicone, AS resin (acrylonitrile-styrene copolymer), polyvinyl chloride, polyvinylidene chloride, polystyrene resin, polyvinyl acetate, ABS resin, polycarbonate resin, vinyl ether, polyacetal (POM), polyamide, polyphenylene ether (PPE), polyaryl ether, polyphenylene sulfide (PPS), polysulfone (PS), polyethersulfone (PES), polyimide (PI), polyamic acid (PAA), polyamideimide acrylic resin, polyimide resin, phenolic resin, polyether ketone resin, polyether nitrile (PEN) resin, and the like.

[0093] In order to improve adhesion to cells, tissues, antibodies, and antigens, the flat substrate may be subjected to a hydrophilic treatment using plasma treatment, corona treatment, or a silane coupling agent to provide a hydroxyl group, a carboxyl group, an amino group, or an ester group on the surface. Of these, it is preferable that the flat substrate has an amino group on the surface. It is also preferable that the substrate surface is provided with a positive charge using a coating agent such as poly-L-lysine, MAS coating, or 3-aminopropyltriethoxysilane.

[0094] The blocking agent of the present invention exerts its performance by coating, with the water-soluble vinyl polymer (A) in the blocking agent, a part or all of a planar substrate on which an antigen or antibody against a target substance is immobilized or a specimen is adhered. The method for immobilizing an antigen or antibody against a target substance on a planar carrier is not particularly limited, but the two may be contacted according to standard methods such as physical adsorption due to hydrophobic-hydrophobic interactions or chemical bonding using a water-soluble carbodiimide-based condensing agent.

[0095] The surface zeta potential of the flat substrate is preferably 0 to +50 mV before the blocking treatment, and is preferably -100 to 0 mV, more preferably -85 to 0 mV, and even more preferably -70 to 0 mV after the blocking treatment. When the surface zeta potential of the flat substrate is within the above range, the blocking agent is adsorbed to the substrate, and an excellent blocking effect can be expected.

[0096] <Blocking method> The blocking method of the present invention includes a step of subjecting a planar substrate to a blocking treatment using a blocking agent. The blocking treatment is performed by immersing a part or the whole of a planar substrate on which an antigen or antibody against a target substance is immobilized or a specimen is attached, in the blocking agent of the present invention. Thereafter, an enzyme-labeled antigen or antibody is added to carry out an antigen-antibody reaction. Furthermore, excess antigen or antibody is washed away, and a color-developing dye is added to develop color. Non-specific adsorption was evaluated by visual observation or absorbance measurement, and adsorption of the color-developing dye to the substrate was evaluated by measuring the transmittance of the substrate. EXAMPLES

[0097] The present invention will be described in more detail below with reference to examples, but the following examples are not intended to limit the scope of the invention. In the examples and comparative examples, "parts" refers to "parts by mass", "mol" refers to the amount of a substance, and "mass%" refers to the proportion of the amount of a substance in the total monomers.

[0098] <Synthesis of Water-soluble Vinyl Polymer (A)> [Production Example 1] A reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux condenser was charged with 100 parts of ethanol, the internal temperature was raised to 75°C, and the atmosphere was thoroughly replaced with nitrogen. A mixture of 1 part of 2,2'-azodiisobutyronitrile (AIBN), 70 parts of N,N-dimethylaminoethyl methacrylate as monomer (a''1), 20 parts of isostearyl acrylate as monomer (b'), and 10 parts of methacrylic acid as monomer (c'), which had been prepared separately, was added dropwise for 2 hours while maintaining the internal temperature at 75°C, and stirring was continued for another 2 hours to obtain a copolymer. After confirming that the conversion rate exceeded 98% by measuring the solid content, 39 parts of 35% hydrogen peroxide solution as a modifier (oxidizing agent) and 200 parts of ion-exchanged water were added, and stirring was continued for another 20 hours while distilling off the ethanol. The reaction product was then cooled and taken out, and the solvent was completely evaporated in a vacuum oven to obtain a water-soluble vinyl polymer (A) P-1. The properties are shown in Table 1.

[0099] [Production Examples 2 to 9, 11 to 13, Comparative Production Examples 1 to 3] Except for changing the composition as shown in Table 1, synthesis was performed in the same manner as in Production Example 1 to obtain water-soluble vinyl polymers (A) P-2 to 9 and P-11 to 16 of Production Examples 2 to 9, 11 to 13 and Comparative Production Examples 1 to 3.

[0100] [Production Example 10] A reaction vessel equipped with a stirrer, a thermometer, a dropping funnel, and a reflux condenser was charged with 100 parts of ethanol, and the internal temperature was raised to 75°C and thoroughly replaced with nitrogen. A mixture of 2 parts of 2,2'-azodiisobutyronitrile, 50 parts of 2-methacryloyloxyethyl phosphorylcholine as monomer (a'4), 20 parts each of butyl methacrylate and methyl methacrylate as monomer (b'), and 10 parts of methacrylic acid as monomer (c'), which had been prepared separately, was added dropwise for 3 hours while maintaining the internal temperature at 75°C, and stirring was continued for another 2 hours. After confirming that the conversion rate exceeded 98% by measuring the solid content, the mixture was cooled and taken out. Thereafter, the solvent was completely evaporated in a vacuum oven to obtain a water-soluble vinyl polymer (A) P-10.

[0101] In addition, 1 g of each of the vinyl polymers (A) P1 to 16 obtained in Production Examples 1 to 13 and Comparative Production Examples 1 to 3 was added to 100 mL of ion-exchanged water at 25° C., stirred to dissolve, and then allowed to stand for 24 hours at 25° C. As a result, neither separation nor precipitation was observed in these resins, indicating that they were completely soluble and water-soluble.

[0102] [Evaluation of physical properties of water-soluble vinyl polymer (A)] <Surface zeta potential> A MAS-coated slide glass (Matsunami Glass Industry Co., Ltd., surface zeta potential +10 mV) was cut to the size of the sample holder (approximately 10 mm x 35 mm) of the flat plate zeta potential measurement cell to prepare a measurement sample. It was immersed in a 1 w / v% aqueous solution of water-soluble vinyl polymer (A) at room temperature for 30 minutes, and then washed three times with pure water. A standard particle aqueous solution was placed in the flat plate zeta potential measurement cell, and the sample holder with the sample set was immersed in it. The zeta potential of the sample surface was obtained by electrophoretic light scattering using an ELSZ-2000Z (Otsuka Electronics Co., Ltd.). The evaluation results are shown in Table 1.

[0103] <ph> The glass electrode of a pH meter (HORIBA) calibrated with a standard solution was immersed in a 1 w / v % aqueous solution of the water-soluble vinyl polymer (A) to measure the pH. The evaluation results are shown in Table 1.

[0104] <Acid value> 1 g of water-soluble vinyl polymer was weighed out and dissolved in 20 g of water, after which one drop of phenolphthalein solution was added. The solution was stirred with a stir bar and a magnetic stirrer, and neutralization titration was performed with 0.1 mol / L potassium hydroxide solution (Kishida Chemical). The acid value was calculated from the titration amount obtained. The evaluation results are shown in Table 1.

[0105] [Table 1]

[0106] The symbols in Table 1 are as follows. DMAEMA: N,N-Dimethylaminoethyl methacrylate VP: 4-vinylpyridine VI: 1-Vinylimidazole MPC: 2-methacryloyloxyethyl phosphorylcholine ISTA: Isostearyl acrylate (LogP=7.64) BMA: Butyl methacrylate (LogP=2.23) MMA: Methyl methacrylate (LogP=0.99) St: styrene (logP=2.67) MAA: methacrylic acid (pKa=4.8) VBA: Vinyl sulfonic acid (pKa=-1.3) HEMP: Mono(2-hydroxyethyl methacrylate) phosphate (pKa=1.6) The logP value of the monomer (b') and the pKa of the monomer (c') were calculated using a program incorporated in the CambridgeSoft system: ChemdrawPro11.0.

[0107] <Preparation of blocking agent> [Examples 1 - 13, Comparative Examples 1 - 3] (Immunostaining method and blocking agent for protein stability evaluation) The water - soluble vinyl - based polymers (A) obtained in Production Examples 1 - 13 and Comparative Production Examples 1 - 3 were each dissolved in PBS to obtain blocking agents for Examples 1 - 13 and Comparative Examples 1 - 3 at a concentration of 1 w / v%. For the water - soluble vinyl - based polymer (A) of Production Example 1, blocking agents at concentrations of 10 w / v% and 0.1 w / v% were also prepared in addition to the 1 w / v% concentration.

[0108] (Blocking agent for ELISA) A blocking agent for ELISA was prepared in the same manner as the preparation of the blocking agent for immunostaining method and protein stability evaluation, except that PBS was changed to PBS - T (0.1% Tween (registered trademark) 20).

[0109] [Performance evaluation of blocking agent] The following evaluations were performed on the obtained blocking agents. The results are shown in Table 2.

[0110] ·Evaluation in immunostaining method <DAB solution> 10 mg of DAB tablets (3,3’ - diaminobenzidine tetrahydrochloride, manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved in 50 mL of 0.05 mol / L Tris - hydrochloric acid buffer, and 10 μL of 30% hydrogen peroxide solution was further added to prepare a staining solution.

[0111] <Non - specific adsorption inhibitory evaluation> A medium was prepared by adding 10% fetal bovine serum (FBS) and 1% penicillin streptomycin (PS) to low glucose-DMEM medium (Sigma-Aldrich) (hereinafter, the above medium will be referred to as FBS-PS-DMEM medium). A polylysine-coated cover glass was placed in a 24-well plate. Human hepatoma cell line HepG2 cells were adjusted to 33,000 cells / mL in FBS-PS-DMEM medium, and then seeded at 1 mL per well. The plate was then cultured stationary for 2 days in a CO2 incubator (37°C, 5% CO2). The medium was then removed, the cells were washed with PBS, and fixed with 4% formaldehyde for 30 minutes. The cells were permeabilized with 0.1% Triton-X for 10 minutes and washed with PBS. HRP-labeled secondary antibody (ab24462: HRP Mouse monoclonal [C6] to C Reactive Protein antibody) was diluted to 8 μg / mL with blocking agent solution and added at 250 μL per well from which PBS had been removed. The wells were left to stand at 37°C for 1 hour and washed with PBS. The above DAB solution was added and left to stand for 30 minutes to allow color development. After washing with PBS, the wells were dehydrated and mounted. After confirming that the mounting medium was dry, the presence or absence of nonspecific adsorption was visually evaluated using a microscope. The evaluation results are shown in Table 2. (Evaluation Criteria) ◎: Very good ○: Good △: Fairly good ×: Poor (dark color due to non-specific adsorption)

[0112] <Prevention of base material discoloration> A MAS-coated slide glass (Matsunami Glass Industry) was immersed in a solution of HRP-labeled secondary antibody (ab24462: HRP Mouse monoclonal [C6] to C Reactive Protein antibody) diluted to 8 μg / mL with 1% blocking agent solution, and allowed to react for 30 minutes at room temperature. After washing three times with PBS, the glass substrate was immersed in the above DAB solution and allowed to react for 10 minutes at room temperature. After washing three times with PBS, the transmittance at a wavelength of 450 nm was measured, and the coloring of the substrate was evaluated. The evaluation results are shown in Table 2. (Evaluation Criteria) ◎: 90% or more (Very good) ○: 85% or more and less than 90% (Good) △: 80% or more and less than 85% (Somewhat good) ×: Less than 80% (Poor: Strong coloring due to non-specific adsorption)

[0113] <Sensitization Effect> For the evaluation, only those with ○ or above in the non-specific adsorption inhibition evaluation of the above immunostaining method were used. A medium was prepared by adding 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) to Low glucose-DMEM medium (manufactured by Sigma-Aldrich) (hereinafter, the above medium is referred to as FBS-PS-DMEM medium). A polylysine-coated cover glass was placed in a 24-well plate. Human hepatocellular carcinoma cell line HepG2 cells were adjusted with FBS-PS-DMEM medium to 33,000 cells / mL and then seeded at 1 mL per well. Thereafter, the plate was cultured in a CO2 incubator (37 °C, 5% CO2) in a static state for 2 days. Thereafter, the medium was removed, washed with PBS, and fixed with 4% formaldehyde for 30 minutes. The HRP-labeled antibody (ab195021: HRP Anti-Glucose Transporter GLUT1 antibody) was diluted to 1 μg / mL with a blocking agent solution, and 250 μL each was added to the wells excluding PBS. It was left standing at room temperature for 1 hour and washed with PBS. The above DAB solution was added and left standing for 5 minutes for color development operation. After washing with PBS, dehydration operation was performed and sealed. After confirming the drying of the embedding agent, the staining intensity was visually evaluated under a microscope. The evaluation results are shown in Table 2. (Evaluation Criteria) ◎: Good (High staining intensity, high sensitization effect) ○: Good (Sensitization effect) △: Somewhat good (Somewhat sensitization effect)

[0114] ·ELISA Evaluation <Preparation of Capture Antibody Diluent> 0.265 g of sodium carbonate was dissolved in 50 mL of ultrapure water (pH 11). Also, 0.210 g of sodium hydrogen carbonate was dissolved in 50 mL of ultrapure water (pH 8). 10 mL of each of these solutions was mixed, and the pH was adjusted to 9.6 while measuring the pH.

[0115] <Evaluation of non-specific adsorption inhibition> The solid-phase antibody (a80-129a: Goat anti-Human Albumin Antibody) was diluted to 3 μg / mL with the capture antibody diluent, and 100 μL was dispensed into each well of a 96-well plate, and then left to stand overnight at 5 ° C. Then, it was washed three times with PBS-T (0.1% Tween (registered trademark) 20). 200 μL of the blocking agent was dispensed into each well and left to stand at 25 ° C. for 2 hours. Then, it was washed three times with PBS-T (0.1% Tween (registered trademark) 20). The HRP-labeled antibody (a80-129P: Goat anti-Human Albumin Antibody HRP Conjugated) was diluted to 15 ng / mL with the blocking agent, and 100 μL was dispensed into each well, and then left to stand at 25 ° C. for 1 hour. Then, it was washed three times with PBS-T (0.05% Tween (registered trademark) 20). ABTS Kit ELISA POD Substrate ABTS Kit (Nacalai Tesque) color development solution, 2,2'-azinodi(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt (ABTS 1 mg / mL) and the substrate solution (hydrogen peroxide / phosphate citrate buffer [pH 4.2]) of the same kit were prepared in a ratio of 10 / 1 (v / v) and 100 μL was dispensed into each well. After stirring, the mixture was left to stand at 25°C for 30 minutes. Then, 100 μL of an oxalic acid solution, which is the reaction stop solution of the same kit, was added to each well. The absorbance at 405 nm was then measured, and the evaluation results are shown in Table 2. (Evaluation Criteria) ◎: Absorbance less than 0.1 (very good) ○: Absorbance 0.1 or more and less than 0.15 (good) △: absorbance 0.15 or more and less than 0.2 (fairly good) ×: absorbance 0.2 or more (poor: non-specific adsorption not suppressed)

[0116] <Sensitization effect> For the evaluation, only those that were rated ◯ or higher in the non-specific adsorption inhibition evaluation by the above-mentioned ELISA were used. The solid-phase antibody (a80-129a: Goat anti-Human Albumin Antibody) was diluted to 3 μg / mL with the capture antibody diluent, and 100 μL was dispensed into each well of a 96-well plate, and the plate was left to stand overnight at 5 ° C. Then, the plate was washed three times with PBS-T (0.1% Tween (registered trademark) 20). 200 μL of the blocking agent was dispensed into each well and the plate was left to stand at 25 ° C. for 2 hours. Then, the plate was washed three times with PBS-T (0.1% Tween (registered trademark) 20). The antigen (010-27601: albumin derived from human serum) was adjusted to 0 to 1200 ng / mL with PBS, and 100 μL was dispensed into each well, and the plate was left to stand at 25 ° C. for 1 hour. Then, the plate was washed three times with PBS-T (0.05% Tween (registered trademark) 20). HRP-labeled antibody (a80-129P: Goat anti-Human Albumin Antibody HRP Conjugated) was diluted to 15 ng / mL with a blocking agent, and 100 μL was dispensed into each well, and then left to stand at 25 ° C for 1 hour. Then, it was washed three times with PBS-T (0.05% Tween (registered trademark) 20). ABTS kit ELISA POD substrate ABTS kit (Nacalai Tesque) color development solution 2,2'-azinodi (3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt (ABTS 1 mg / mL) and the substrate solution of the same kit (hydrogen peroxide water / phosphate citrate buffer [pH 4.2]) were prepared at a ratio of 10 / 1 (v / v), and 100 μL was dispensed into each well. After stirring, it was left to stand at 25 ° C for 30 minutes. Then, 100 μL of oxalic acid aqueous solution, which is the reaction stop solution of the same kit, was added to each well. The absorbance at 405 nm was then measured. Sensitization was evaluated from the absorbance when the antigen concentration was 1200 ng / mL. The evaluation results are shown in Table 2. (Evaluation Criteria) ◎: Absorbance 1.2 or more (very good, high sensitizing effect) ○: absorbance 1.1 or more but less than 1.2 (good, has a sensitizing effect) △: absorbance less than 1.1 (slightly good, slight sensitizing effect)

[0117]

Table 2

[0118] (3) Evaluation of protein stability

[0119] <Stability test of antibody> <Preparation of TMBz chromogenic solution> Using a peroxidase chromogenic kit (manufactured by Sumitomo Bakelite), 10 mL of the chromogenic agent (3,3’,5,5’-tetramethylbenzidine (TMBZ) solution) and 100 μL of the substrate solution (hydrogen peroxide solution) were mixed to obtain the TMBz chromogenic solution.

[0120] The HRP-labeled secondary antibody (ab24462: HRP Mouse monoclonal [C6] to C Reactive Protein antibody) was diluted with the blocking agent solution to 80 ng / mL and stored at 40 °C for 14 days. After adding 100 μL of the solution to a 96-well plate, 100 μL of the TMBz chromogenic solution was added and left standing at 37 °C for 30 minutes. Then, 100 μL of the stop solution was added, and the absorbance at 450 nm (reference: 620 nm) was measured. The ratio when the absorbance on day 0 was set to 1 was calculated and evaluated. The evaluation results are shown in Table 3. (Evaluation criteria) ◎: 80% or more (very good) ○: 70% or more and 80% or less (good) △: 50% or more and 70% or less (fairly good) ×: 50% or less (poor)

[0121] <Stability test of lysozyme> Lysozyme was diluted with a blocking agent to 0.15 mg / mL and allowed to stand at 40°C for 6 weeks. 200 μL was added to a 96-well plate, and then 10 μL of the substrate Micrococcus luteus suspension (1 mg / mL, PBS pH 7.4) was added, and the change in optical density (OD value) at a wavelength of 450 nm over time was immediately measured at room temperature. The ratio of the slopes obtained at this time was defined as the enzyme activity. The percentage of remaining activity was calculated when the activity of the sample stored at 4°C was set to 1. The evaluation results are shown in Table 3. (Evaluation Criteria) ◎: 75% or more (very good) ○: 60% or more and 75% or less (good) △: 50% to 60% (fairly good) ×: 50% or less (defective)

[0122] [Table 3]

[0123] <Result> The results in Tables 2 and 3 reveal that the blocking agents of the present invention exhibit excellent non-specific adsorption suppression effects, protein stabilization effects, and sensitization effects. On the other hand, the additives of Comparative Examples 1 to 3 were inferior to the blocking agents of the Examples in terms of blocking properties, substrate coloration prevention properties, sensitization properties, and protein stability.

[0124] As described above, the blocking agent of the present invention can prevent non-specific adsorption and substrate coloration that may lead to erroneous judgments when performing in vitro diagnosis such as ELISA, immunohistochemical staining, and Western blotting. In addition, since the blocking agent of the present invention has a protein stabilizing effect and a sensitizing effect, it is possible to prevent denaturation of antibodies and the like used in in vitro diagnosis and obtain accurate results. In addition, since the blocking agent of the present invention is made of a non-biological substance, its performance is easily stabilized, and stable in vitro diagnosis can be performed.

[0125] The blocking agent of the present invention has high adhesion to a flat substrate, and unlike conventional blocking agents, can stably exhibit excellent non-specific adsorption prevention ability, slide coloring suppression ability, protein stabilization ability, and color development sensitization effect in immunostaining. By using this blocking agent, it becomes possible to perform in vitro diagnosis such as ELISA, immunohistochemical staining, and Western blotting with high sensitivity.< / ph>

Claims

1. A blocking agent for preventing non-specific adsorption in an antigen-antibody reaction carried out on a planar substrate, Contains a water-soluble vinyl polymer (A), The water-soluble vinyl polymer (A) contains the following structural units (a), (b), and (c), The blocking agent has a content of the structural unit (c) of 5 to 35% by mass relative to 100% by mass of the water-soluble vinyl polymer (A). Structural unit (a): structural unit having a zwitterionic structure Structural unit (b): a structural unit derived from a monomer (b') having a partition coefficient Log P of 0.8 to 10 Structural unit (c): a structural unit derived from a monomer (c') having a pKa in water of -3 to 5 (excluding those having a zwitterionic structure)

2. 2. The blocking agent according to claim 1, wherein the water-soluble vinyl polymer (A) has an acid value of 20 to 250 mg KOH / g.

3. 3. The blocking agent according to claim 1 or 2, wherein a slide glass having a surface zeta potential of +10 mV has a surface zeta potential of −100 to 0 mV when covered with a 1 w / v % aqueous solution of the water-soluble vinyl polymer (A).

4. 3. The blocking agent according to claim 1, wherein the pH of a 1 w / v % aqueous solution of the water-soluble vinyl polymer (A) is 3 to 7.

5. 3. The blocking agent according to claim 1, wherein the structural unit (c) has one or more functional groups selected from the group consisting of a carboxy group, a sulfo group, and a phosphate group.

6. 3. The blocking agent according to claim 1 or 2, wherein the structural unit (a) has one or more zwitterionic structures selected from the group consisting of phosphorylcholine, sulfobetaine, carbobetaine, and amine oxide.

7. 7. The blocking agent according to claim 6, wherein the structural unit (a) has one or more zwitterionic structures selected from the group consisting of the following formulas (1) to (7): General formula (1) 【Chemistry 1】 General formula (2) 【Chemistry 2】 General formula (3) 【Chemistry 3】 General formula (4) 【Chemistry 4】 General formula (5) 【Chemistry 5】 General formula (6) 【Chemistry 6】 General formula (7) 【Chemistry 7】 [In general formulas (1) to (7), X is an oxygen atom or NH; Y is the COO － Or SO ３ － , R ２ , R １６ , R ２７ each independently represents an alkylene group having 1 to 6 carbon atoms, R ３ , R ４ , R １７ , R １８ , R ２９ , R ３０ , R ３１ each independently represents an alkyl group having 1 to 4 carbon atoms, R ５ , R ２８ each independently represents an alkylene group having 1 to 4 carbon atoms, R ７ , R ２０ each independently represents a hydrogen atom or a methyl group, R ８ is an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms, R ９ ~R １３ Four of them are R ２１ ~R ２５ four of the groups are independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R １４ represents an alkylene group having 1 to 6 carbon atoms or a hydroxyalkylene group having 1 to 6 carbon atoms, R ９ ~R １３ One of them, R ２１ ~R ２５ one of the groups represents a bonding site with the main chain of the vinyl polymer, * indicates the bonding position with the main chain of the vinyl polymer.

8. 3. The blocking agent according to claim 1, wherein the content of the structural unit (a) is 35 to 85% by mass in 100% by mass of the water-soluble vinyl polymer (A).

9. 3. The blocking agent according to claim 1, wherein the content of the structural unit (b) is 10 to 50 mass % in 100 mass % of the water-soluble vinyl polymer (A).

10. 3. The blocking agent according to claim 1, wherein the water-soluble vinyl polymer (A) has a mass average molecular weight of 2,000 to 1,000,000.

11. 3. The blocking agent according to claim 1, further comprising water, wherein the concentration of the water-soluble vinyl polymer (A) is 0.01 to 30.0 w / v %.

12. A kit comprising the blocking agent according to claim 1 or 2 and a planar substrate.

13. The kit according to claim 12, wherein the surface zeta potential of the planar substrate is 0 to +50 mV before the blocking treatment.

14. A blocking method comprising the step of subjecting a flat substrate to a blocking treatment with the blocking agent according to claim 1 or 2.

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