JP2009128093A - Method of detecting physiological active substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a physiological active substance detection method which is easily operated and capable of measurements in a visible region. <P>SOLUTION: An ethylene-based unsaturated polymerizable monomer having an alkylene glycol residue, an ethylene-based unsaturated polymerizable monomer having a functional group for immobilizing physiological active substances, and an ethylene-based unsaturated polymerizable monomer having a crosslinkable functional group are copolymerized to form a high-molecular substance, and an insoluble carrier has the high-molecular substance in its surface. An antibody is immobilized in the insoluble carrier to bring a biologically active substance to be an antigen into reaction with the antibody. An enzyme is introduced to a reacted antibody-antigen complex to make a color-developing substrate develop a color by the action of the enzyme. The contained situation of the physiological active substance in an object to be detected is determined on the basis of the degree of color development in the physiological active substance detection method. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for detecting a physiologically active substance by immobilizing an antibody on a predetermined carrier surface, and determining the presence and amount of the physiologically active substance as an antigen specifically bound to the immobilized antibody in the visible light region. It is.

  Attempts to decipher the genetic activity and the molecular processes of drug effects at the molecular level have traditionally focused on genomics, but proteomics is more detailed about the biological functions of cells. Provide information. Proteomics involves the qualitative and quantitative measurement of gene activity by detecting and quantifying expression at the protein level, rather than at the gene level. It also includes studies of events that are not encoded by genes such as post-translational modifications of proteins and interactions between proteins.

  Now that the structure of the genome, which is the “blueprint of life”, has been clarified and a large amount of genome information has become available, proteomics research has become increasingly popular, and as a result, rapid detection of bioactive substances has been accelerated. There is a need for higher efficiency (high throughput). A DNA chip has been developed and put into practical use as a molecular array for this purpose. On the other hand, protein chips have been proposed for the detection of the most complex and highly diverse proteins in biological functions, and research has been promoted in recent years. The protein chip is a general term for a protein or a molecule that captures the protein immobilized on the surface of the chip (micro substrate).

    However, in the detection of a physiologically active substance using a protein chip, it is generally necessary to use an expensive fluorescent reagent for detection, and an expensive detection device dedicated to microarrays is required for detection, and its use is also part of the research field. It is limited to research institutes, and it has not yet been used in clinical inspection fields and food inspection fields.

One means for solving the above problem is detection by visual recognition. For example, Patent Document 1 discloses a method for detecting a gene visually. The method described in Patent Document 1 is a method of performing gene amplification by LAMP method and visualizing using an intercalator such as SYBR Green I. However, this method is intended for genes and is a protein. A physiologically active substance such as cannot be detected.
JP 2004-154008 A

    An object of the present invention is to provide a method for detecting a quantitatively active physiologically active substance that is easy to operate and does not use a special instrument or the like and can be measured in the visible region.

（式中Ｒ₁は水素原子またはメチル基を示し、Ｒ_４は水素原子または炭素数１〜２０のアルキル基を示す。Ｘは炭素数１〜１０のアルキレングリコール残基を示し、ｐは１〜１００の整数を示す。繰り返されるＸは、同一であっても、または異なるアルキレングリコール基の連鎖であってもよい。）
（８）前記アルキレングリコール残基を有するエチレン系不飽和重合性モノマー（Ａ）がメトキシポリエチレングリコール（メタ）アクリレートである（７）記載の生理活性物質の検出方法、
（９）前記メトキシポリエチレングリコール（メタ）アクリレートのエチレングリコールの平均連鎖が３〜１００である（８）記載の生理活性物質の検出方法、
（１０）生理活性物質を固定化する官能基を有するエチレン系不飽和重合性モノマー（Ｂ）の官能基がアルデヒド基、活性エステル、エポキシ基、ビニルスルホン基、ビオチンから選ばれる少なくとも一つの官能基である（１）〜（９）いずれか記載の生理活性物質の検出方法、
（１１）生理活性物質を固定化する官能基を有するエチレン系不飽和重合性モノマー（Ｂ）が下記の一般式［２］で表される活性エステルを有するモノマーである（１）〜（９）いずれか記載の生理活性物質の検出方法、

（式中Ｒ_２は水素原子またはメチル基を示し、Ｙは炭素数１〜１０のアルキレングリコール残基またはアルキル基を示す。Ｗは活性エステル基を示す。ｑは１〜２０の整数を示す。ｑが２以上２０以下の整数である場合、繰り返されるＹは、それぞれ同一であっても、または異なるアルキレングリコール残基の連鎖であってもよい。）
（１２）前記活性エステルがｐ−ニトロフェニルエステル又はＮ−ヒドロキシスクシンイミドエステルである（１０）または（１１）記載の生理活性物質の検出方法、
（１３）架橋可能な官能基を有するエチレン系不飽和重合性モノマー（Ｃ）の架橋可能な官能基がアルコキシシリル、エポキシ、及び（メタ）アクリルから選ばれる少なくとも一つの官能基である（１）〜（１２）いずれか記載の生理活性物質の検出方法、
（１４）架橋可能な官能基を有するエチレン系不飽和重合性モノマー（Ｃ）が下記の一般式［３］で表されるアルコキシシリルを有するモノマーである（１）〜（１２）いずれか記載の生理活性物質の検出方法、

（式中Ｒ_３は水素原子またはメチル基を示し、Ｚは炭素数１〜２０のアルキル基を示す。Ａ_１、Ａ_２、Ａ_３のうち、少なくとも１個は加水分解可能なアルコキシ基であり、その他はアルキル基を示す。）
（１５）発色した色素が抗体または抗原およびその近傍の担体表面に付着し、色素の付着の度合いを色の濃さとして測定することを特徴とする（１）〜（１４）いずれか記載の生理活性物質の検出方法、
（１６）発色の度合いを吸光により測定することを特徴とする（１）〜（１５）いずれか記載の生理活性物質の検出方法、
（１７）前記抗体が担体表面にスポット状に固定化されている（１）〜（１６）いずれか記載の生理活性物質の検出方法、
（１８）複数種の抗体のスポットが担体表面の同一区画中に存在している（１７）記載の生理活性物質の検出方法、
（１９）担体の形状がスライドグラス状、９６穴プレート状、３８４穴プレート状、１５３６穴プレート状、マイクロ流路、ビーズ、チューブ、又は容器及びそれらの複合体よりなる群より選択された少なくとも１つである（１）〜（１８）いずれか記載の生理活性物質の検出方法、
（２０）担体の材質がプラスチックである（１）〜（１９）いずれか記載の生理活性物質の検出方法、
（２１）プラスチックがポリカーボネート、ポリエチレン、ポリプロピレン、ポリスチレン、飽和環状ポリオレフィン、ポリペンテン、ポリアミド、及びそれらの共重合体よりなる群より選択された少なくとも１種である（２０）記載の生理活性物質の検出方法、
である。 That is, the present invention
(1) Ethylene unsaturated polymerizable monomer (A) having an alkylene glycol residue, ethylene unsaturated polymerizable monomer (B) having a functional group for immobilizing a physiologically active substance, and ethylene having a crosslinkable functional group Using an insoluble carrier having a polymer material copolymerized with an unsaturated polymerizable monomer (C) on the surface,
(A) producing an antibody-immobilized carrier by immobilizing an antibody on the surface of the insoluble carrier;
(B) adding a solution containing a physiologically active substance as an antigen on the antibody-immobilized carrier and reacting with the antibody;
(C) binding an enzyme-containing substance to the reacted antibody-antigen complex;
(D) adding a solution containing a chromogenic substrate and causing the chromogenic substrate to develop color by the reaction of the enzyme;
A method for detecting a physiologically active substance, wherein the content of the physiologically active substance in the solution in step (b) is determined according to the degree of color development,
(2) In addition to the above steps,
(B-2) a step of further adding a solution containing a secondary antibody to the reacted antibody-antigen complex and reacting with the complex;
A method for detecting a physiologically active substance according to (1),
(3) In step (b-2), the secondary antibody is preliminarily labeled with biotin, and the solution containing the enzyme-labeled avidin in step (c) is brought into contact with the carrier surface to form an antibody-antigen complex. The method for detecting a physiologically active substance according to (2), wherein the enzyme is labeled;
(4) The method for detecting a physiologically active substance according to any one of (1) to (3), wherein the enzyme labeled with the antibody-antigen complex is an oxidation or reductase enzyme,
(5) The method for detecting a physiologically active substance according to any one of (1) to (4), wherein the enzyme to be labeled is peroxidase or alkaline phosphatase,
(6) The method for detecting a physiologically active substance according to any one of (1) to (5), wherein the antibody is covalently bonded to and immobilized on the surface of the carrier in step (a).
(7) Detection of a physiologically active substance according to any one of (1) to (6), wherein the ethylenically unsaturated polymerizable monomer (A) having an alkylene glycol residue is a monomer represented by the following general formula [1] Method,

(Wherein R ₁ represents a hydrogen atom or a methyl group, R ₄ represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms, X represents an alkylene glycol residue having 1 to 10 carbon atoms, and p represents 1 to Represents an integer of 100. The repeated Xs may be the same or a chain of different alkylene glycol groups.
(8) The method for detecting a physiologically active substance according to (7), wherein the ethylenically unsaturated polymerizable monomer (A) having an alkylene glycol residue is methoxypolyethylene glycol (meth) acrylate,
(9) The method for detecting a physiologically active substance according to (8), wherein the average chain of ethylene glycol in the methoxypolyethylene glycol (meth) acrylate is 3 to 100,
(10) At least one functional group in which the functional group of the ethylenically unsaturated polymerizable monomer (B) having a functional group for immobilizing a physiologically active substance is selected from an aldehyde group, an active ester, an epoxy group, a vinyl sulfone group, and biotin The method for detecting a physiologically active substance according to any one of (1) to (9),
(11) The ethylenically unsaturated polymerizable monomer (B) having a functional group for immobilizing a physiologically active substance is a monomer having an active ester represented by the following general formula [2] (1) to (9) A method for detecting a physiologically active substance according to any one of the above,

(In the formula, R ₂ represents a hydrogen atom or a methyl group, Y represents an alkylene glycol residue or an alkyl group having 1 to 10 carbon atoms, W represents an active ester group, and q represents an integer of 1 to 20.) When q is an integer of 2 or more and 20 or less, each repeated Y may be the same or a chain of different alkylene glycol residues.
(12) The method for detecting a physiologically active substance according to (10) or (11), wherein the active ester is p-nitrophenyl ester or N-hydroxysuccinimide ester,
(13) The crosslinkable functional group of the ethylenically unsaturated polymerizable monomer (C) having a crosslinkable functional group is at least one functional group selected from alkoxysilyl, epoxy, and (meth) acrylic (1) To (12) a method for detecting a physiologically active substance according to any one of
(14) The ethylenically unsaturated polymerizable monomer (C) having a crosslinkable functional group is a monomer having an alkoxysilyl represented by the following general formula [3] (1) to (12) A method for detecting a physiologically active substance,

(In the formula, R ₃ represents a hydrogen atom or a methyl group, and Z represents an alkyl group having 1 to 20 carbon atoms. At least one of A ₁ , A ₂ , and A ₃ is a hydrolyzable alkoxy group. And others represent alkyl groups.)
(15) The physiology according to any one of (1) to (14), wherein the colored dye adheres to the antibody or antigen and the nearby carrier surface, and the degree of dye adhesion is measured as the color intensity. Active substance detection method,
(16) The method for detecting a physiologically active substance according to any one of (1) to (15), wherein the degree of color development is measured by absorption,
(17) The method for detecting a physiologically active substance according to any one of (1) to (16), wherein the antibody is immobilized in a spot shape on the surface of a carrier,
(18) The method for detecting a physiologically active substance according to (17), wherein spots of a plurality of types of antibodies are present in the same compartment on the surface of the carrier,
(19) The shape of the carrier is at least one selected from the group consisting of a glass slide, a 96-hole plate, a 384-hole plate, a 1536-hole plate, a microchannel, a bead, a tube, or a container and a complex thereof. The method for detecting a physiologically active substance according to any one of (1) to (18),
(20) The method for detecting a physiologically active substance according to any one of (1) to (19), wherein the carrier material is plastic.
(21) The method for detecting a physiologically active substance according to (20), wherein the plastic is at least one selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefin, polypentene, polyamide, and copolymers thereof. ,
It is.

  According to the method for detecting a physiologically active substance of the present invention, it is possible to detect a physiologically active substance in the visible region without using a special reading device or the like without using a fluorescent dye. Furthermore, it is possible not only to determine the presence or absence of a physiologically active substance, but also to quantify it.

    The method for detecting a physiologically active substance of the present invention comprises an ethylenically unsaturated polymerizable monomer (A) having an alkylene glycol residue, an ethylenically unsaturated polymerizable monomer (B) having a functional group for immobilizing the physiologically active substance, and An antibody was immobilized on an insoluble carrier having a polymer material copolymerized with an ethylenically unsaturated polymerizable monomer (C) having a crosslinkable functional group on the surface, and a physiologically active substance serving as an antigen was reacted and reacted. A substance containing an enzyme is bound to an antibody-antigen complex, a chromogenic substrate is colored by an enzymatic reaction, and the presence or absence of a physiologically active substance to be detected in a sample is determined and quantified in a visible region.

    The polymer substance is a polymer having both the property of suppressing nonspecific adsorption of a physiologically active substance, the property of immobilizing the physiologically active substance, and the property of crosslinking the polymer main chains, and the alkylene glycol residue is physiological. It plays the role which suppresses nonspecific adsorption | suction of an active substance, and the functional group which fix | immobilizes a bioactive substance plays the role which immobilizes a bioactive substance.

    The structure of the ethylenically unsaturated polymerizable monomer (A) having an alkylene glycol residue used for the polymer substance present on the carrier surface of the present invention is not particularly limited, but is represented by the general formula [1] ( A compound composed of a chain of a (meth) acrylic group and an alkylene glycol residue X having 1 to 10 carbon atoms is preferable.

    The alkylene glycol residue X in the formula has 1 to 10 carbon atoms, more preferably 1 to 6, more preferably 2 to 4, still more preferably 2 to 3, and most preferably 2. It is. The repeating number p of the alkylene glycol residue X is an integer of 1 to 100, more preferably an integer of 2 to 100, still more preferably an integer of 2 to 95, and most preferably an integer of 20 to 90. is there. When the number of repeats is 2 or more and 100 or less, the carbon number of the alkylene glycol residue X to be repeated may be the same or different.

    Examples of the ethylenically unsaturated polymerizable monomer (A) having an alkylene glycol residue include methoxypolyethylene glycol (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, and 2-hydroxy. (Meth) acrylates of mono-substituted hydroxyl groups such as butyl (meth) acrylate, glycerol mono (meth) acrylate, (meth) acrylate having polypropylene glycol as a side chain, 2-methoxyethyl (meth) acrylate, 2-ethoxy Examples include ethyl (meth) acrylate, methoxydiethylene glycol (meth) acrylate, ethoxydiethylene glycol (meth) acrylate, and ethoxypolyethylene glycol (meth) acrylate. Methoxy polyethylene glycol methacrylate is preferably from sexual.

    Examples of the functional group of the ethylenically unsaturated polymerizable monomer (B) having a functional group for immobilizing a physiologically active substance used in the present invention include a chemically active group, a receptor group, and a ligand group. It is not limited to. Specific examples include aldehyde groups, active esters, epoxy groups, vinyl sulfone groups, biotin, thiol groups, amino groups, isocyanate groups, isothiocyanate groups, hydroxyl groups, acrylate groups, maleimide groups, hydrazide groups, azide groups, Examples include, but are not limited to, amide groups, sulfonate groups, streptavidin, and metal chelates. Among these, an aldehyde group, an active ester, an epoxy group, and a vinyl sulfone group are preferable from the viewpoint of reactivity with an amino group contained in a large amount in the physiologically active substance, and biotin having a high binding constant with the physiologically active substance is preferable. Of these, active esters are most preferable from the viewpoint of storage stability of the monomer.

    The ethylenically unsaturated polymerizable monomer (B) having a functional group for immobilizing a physiologically active substance used in the present invention is not particularly limited in structure, but (meth) acryl represented by the following general formula [2] A group in which a group and an active ester group are bonded via a chain of an alkylene glycol residue having 1 to 10 carbon atoms or an alkyl group is preferable.

    In the formula [2], the alkylene glycol residue Y has 1 to 10 carbon atoms, more preferably 1 to 6, more preferably 2 to 4, still more preferably 2 to 3, Preferably it is 2. The repeating number q of the alkylene glycol residue Y is an integer of 1 to 20, more preferably an integer of 2 to 18, still more preferably an integer of 3 to 16, and most preferably an integer of 4 to 14. . When the number of repetitions is 2 or more and 20 or less, the carbon number of the alkylene glycol residue to be repeated may be the same or different.

    The “active ester group” used in the present invention is an ester group having a highly acidic electron-withdrawing group in one of the substituents of the ester group and activated for nucleophilic reaction, ie, the reaction activity. As meaning a high ester group, it is commonly used in the fields of various chemical syntheses such as polymer chemistry and peptide synthesis. In practice, phenol esters, thiophenol esters, N-hydroxyamine esters, esters of heterocyclic hydroxy compounds, etc. are known as active ester groups having much higher activity than alkyl esters and the like. .

    Examples of such active ester groups include p-nitrophenyl active ester group, N-hydroxysuccinimide active ester group, succinimide active ester group, phthalimide active ester group, 5-norbornene-2,3-dicarboxy Although an imide active ester group etc. are mentioned, a p-nitrophenyl active ester group or an N-hydroxysuccinimide active ester group is preferable, and a p-nitrophenyl active ester group is most preferable.

    The desirable composition ratio of the ethylenically unsaturated polymerizable monomer (B) having a functional group for immobilizing the physiologically active substance used in the present invention is 1 to 50 mol%, preferably 1 to 30 mol%, most preferably 1 to 1 mol%. 20 mol%.

    The ethylenically unsaturated polymerizable monomer (C) having a crosslinkable functional group used in the present invention is not particularly limited as long as the reaction of the crosslinkable functional group does not proceed during the synthesis of the polymer substance. Absent.

    Examples of functional groups that can be cross-linked include functional groups that generate silanol groups by hydrolysis, epoxy groups, (meth) acryl groups, glycidyl groups, and the like. A functional group, an epoxy group, or a glycidyl group is preferable, and a functional group that generates a silanol group by hydrolysis is preferable because it can be cross-linked at a lower temperature.

    The functional group that generates a silanol group by hydrolysis is a group that readily undergoes hydrolysis and forms a silanol group when contacted with water. For example, a halogenated silyl group, an alkoxysilyl group, a phenoxysilyl group, an acetoxysilyl group Etc. An alkoxysilyl group, a phenoxysilyl group, and an acetoxysilyl group are preferable because they do not contain a halogen. Among them, an alkoxysilyl group is most preferable because a silanol group is easily generated.

  The ethylenically unsaturated polymerizable monomer having a functional group that generates a silanol group by hydrolysis has a general formula in which a (meth) acryl group and an alkoxysilyl group are bonded directly or via an alkyl chain having 1 to 20 carbon atoms. It is preferable that it is an ethylenically unsaturated polymerizable monomer represented by 3].

    Examples of the ethylenically unsaturated polymerizable monomer containing an alkoxysilyl group include 3- (meth) acryloxypropenyltrimethoxysilane, 3- (meth) acryloxypropylbis (trimethylsiloxy) methylsilane, and 3- (meth). Acryloxypropyldimethylmethoxysilane, 3- (meth) acryloxypropyldimethylethoxysilane, 3- (meth) acryloxypropylmethyldimethoxysilane, 3- (meth) acryloxypropylmethyldiethoxysilane, 3- (meth) acryl Roxypropyltrimethoxysilane, 3- (meth) acryloxypropyltriethoxysilane, 3- (meth) acryloxypropyltris (methoxyethoxy) silane, 8- (meth) acryloxyoctanyltrimethoxysilane, 11- (meth) A And the like Lilo carboxymethyl undecene sulfonyl trimethoxysilane the (meth) acryloxy alkyl silane compound. Of these, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropyldimethylmethoxysilane, and 3-methacryloxypropyldimethylethoxysilane have an alkylene glycol residue. This is preferable from the viewpoint of excellent copolymerizability with the monomer and easy availability. These ethylenically unsaturated polymerizable monomers having an alkoxysilyl group are used alone or in combination of two or more.

    The desirable composition ratio of the ethylenically unsaturated polymerizable monomer (C) having a crosslinkable functional group used in the present invention is 1 to 20 mol%, preferably 2 to 15 mol%, most preferably 2 to 10 mol%. .

    The polymer substance used in the present invention is an ethylene unsaturated group having another group in addition to an alkylene glycol residue, an ethylenically unsaturated polymerizable monomer having a functional group capable of immobilizing a physiologically active substance and a crosslinkable functional group. A polymerizable monomer may be included. For example, an ethylenically unsaturated polymerizable monomer (D) having an alkyl group may be copolymerized. As the ethylenically unsaturated polymerizable monomer (D) having an alkyl group, n-butyl methacrylate or n-dodecyl methacrylate or n-octyl methacrylate is preferred.

    The method for synthesizing the polymer substance of the present invention is not particularly limited, but for ease of synthesis, the ethylenically unsaturated polymerizable monomer (A) having at least an alkylene glycol residue and a physiologically active substance are immobilized. Radical polymerization of a mixture containing an ethylenically unsaturated polymerizable monomer (B) having a functional group and an ethylenically unsaturated polymerizable monomer (C) having a crosslinkable group in a solvent in the presence of a polymerization initiator. preferable.

    Any solvent may be used as long as it dissolves each ethylenically unsaturated polymerizable monomer, and examples thereof include methanol, ethanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, and chloroform. These solvents are used alone or in combination of two or more. When the polymer substance is applied to a plastic carrier, ethanol and methanol are preferable because they do not denature the carrier.

    The polymerization initiator may be any ordinary radical initiator such as 2,2′-azobisisobutylnitrile (hereinafter referred to as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), and the like. Examples thereof include organic peroxides such as azo compounds, benzoyl peroxide, and lauryl peroxide.

    The chemical structure of the polymer material of the present invention is such that each ethylenically unsaturated polymerizable monomer having at least an alkylene glycol residue, a functional group for immobilizing a physiologically active substance, and a crosslinkable functional group is copolymerized. For example, the bonding method may be random, block, graft, or the like.

  The molecular weight of the polymer substance of the present invention is preferably 5,000 or more and more preferably 10,000 or more because the polymer substance and the unreacted ethylenically unsaturated polymerizable monomer can be easily separated and purified.

    The polymer substance of the present invention easily imparts the property of suppressing nonspecific adsorption of a physiologically active substance and the property of immobilizing a specific physiologically active substance by coating the carrier surface with the polymer substance. Is possible. Furthermore, since it has the property of cross-linking polymer main chains, it can be cross-linked after coating the carrier surface. Thereby, insolubility can be imparted to the polymer on the carrier, and signal degradation due to substrate cleaning can be reduced.

    For coating the carrier surface with a polymer substance, for example, a polymer solution in which a polymer substance is dissolved in an organic solvent so as to have a concentration of 0.05 to 10% by weight is prepared, and a known method such as immersion or spraying is used. After coating on the surface of the substrate, the drying is performed at room temperature or under heating. Thereafter, the main chains of the polymer are cross-linked by an arbitrary method according to the cross-linkable functional group. When the crosslinkable functional group is a functional group that generates a silanol group by hydrolysis, a mixed solution containing water in an organic solvent may be used. Hydrolysis is caused by the contained water, silanol groups are generated in the synthetic polymer, and the main chains are bonded to each other by heating, so that the polymer substance becomes insoluble.

    When the water content is low, silanol groups are not sufficiently generated and the cross-linking is weakened. On the other hand, when the water content increases, the polymer substance may become insoluble in the solvent. Theoretically, it is sufficient if the water necessary for generating silanol groups by hydrolysis is contained, but considering the ease of preparing the solution, the water content is about 0.01 to 15% by weight. Is preferred.

    Examples of organic solvents include ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, n-pentanol, cyclohexanol, and other alcohols, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, acetone, methyl acetate, ethyl acetate, Examples thereof include butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, and cyclohexanone. These solvents are used alone or in combination of two or more. Among these, alcohols such as ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, n-pentanol, and cyclohexanol are preferable because they do not denature the plastic carrier and can be easily dried. Moreover, when hydrolyzing a high molecular substance in a solution, since it mixes with water in arbitrary ratios, it is preferable.

    In the step of applying the solution in which the polymer material of the present invention is dissolved to the carrier surface and then drying, the silanol groups in the polymer material are dehydrated and condensed with silanol groups, hydroxyl groups, amino groups, etc. in other polymer materials. To form a crosslink. Further, when a hydroxyl group, a carbonyl group, an amino group or the like is present on the support surface, it can be similarly dehydrated and condensed and chemically bonded to the support surface. Since the covalent bond formed by the dehydration condensation of the silanol group has the property of being hardly hydrolyzed, the polymer substance coated on the surface of the carrier is not easily dissolved or detached from the carrier. The dehydration condensation of silanol groups is promoted by heat treatment. Heat treatment is preferably performed within a temperature range where the polymer substance is not denatured by heat, for example, at 60 to 120 ° C. for 5 minutes to 24 hours.

  The material for the bioassay carrier used in the present invention may be glass, plastic, metal, or the like. From the viewpoint of easy surface treatment and mass productivity, plastic is preferable, and thermoplastic resin is more preferable.

    As the thermoplastic resin, those having a small amount of fluorescence generation are preferable, for example, linear polyolefins such as polyethylene and polypropylene, cyclic polyolefins, fluorine-containing resins, etc. are preferably used, heat resistance, chemical resistance, low fluorescence, It is more preferable to use a saturated cyclic polyolefin that is particularly excellent in moldability. Here, the saturated cyclic polyolefin refers to a saturated polymer obtained by hydrogenating a polymer having a cyclic olefin structure or a copolymer of a cyclic olefin and an α-olefin.

    In order to enhance the adhesion between the carrier surface and the polymer material coated on the surface, it is preferable to activate the carrier surface. As a means for activation, there are a method of plasma treatment under an oxygen atmosphere, an argon atmosphere, a nitrogen atmosphere, an air atmosphere and the like, and a method of treatment with an excimer laser such as argon fluoride and krypton fluoride. A method of plasma treatment in an oxygen atmosphere is preferred.

    By applying the polymer substance of the present invention to a carrier, a physiologically active substance-immobilized carrier in which non-specific adsorption of the physiologically active substance on the carrier can be easily produced. Furthermore, the polymer substance on the carrier can be rendered insoluble by crosslinking the polymer substance. From these facts, the carrier coated with the polymer substance can be suitably used for biochips and ELISA plates.

    The shape of the carrier used in the present invention is not particularly limited, but it is a substrate shape typified by a slide glass, a microtiter plate shape typified by 96 or 384 holes, a microchannel, or a bead shape. Tube-like or containers and composites thereof.

In the present invention, when the antibody is immobilized on a carrier, a method of spotting a liquid in which the antibody is dissolved or dispersed is preferred.
The pH of the liquid in which the antibody is dissolved or dispersed is preferably 8 to 10, and more preferably pH 9.0 to 9.9. After immobilization, it is preferable to wash with pure water or a buffer solution in order to remove the physiologically active substance that has not been immobilized.
In addition, it is preferable that the antibody is immobilized in a spot shape on the surface of the carrier, and it is preferable that spots of a plurality of types of antibodies exist in the same compartment on the surface of the carrier.

A solution containing a physiologically active substance serving as an antigen is added to the carrier on which the antibody is immobilized, and the solution is developed on the carrier, whereby the physiologically active substance is captured by the immobilized antibody.
In the present invention, the pH of the liquid in which the physiologically active substance is dissolved or dispersed is preferably 6.0 to 7.6. If the pH is less than 6.0, the physiologically active substance detected by the acid may be denatured, which is not preferable. Moreover, when the pH exceeds 7.6, the physiologically active substance detected by alkali may be denatured, which is not preferable.

    After completion of the antigen-antibody reaction, a substance containing the enzyme is bound to introduce the enzyme into the antibody-antigen complex. The method of introducing the enzyme is, for example, a method using an enzyme-labeled secondary antibody, or a so-called biotin-avidin reaction in which a biotin-labeled secondary antibody is bound, followed by addition of a solution containing the enzyme-labeled avidin. The method to use etc. are mentioned. As the enzyme to be introduced, an enzyme that develops a color reagent is preferably selected, and depending on what is used as the color reagent, peroxidase and alkaline phosphatase are conventionally used as color reagent enzymes, and these are not used. Considering the availability, it is preferable.

    Finally, the chromogenic substrate is colored by the enzyme introduced above. As the coloring reagent, NBT / BICP coloring reagent often used for color development of a membrane such as Western blot, TMBZ, OPD, etc. often used for coloring in the field of ELISA can be used.

    The degree of color development by the color developing reagent depends on the amount of enzyme introduced into the antibody-antigen complex, that is, the amount of antigen. The NBT / BICP coloring reagent adheres to the antibody-antigen complex or the immobilized antibody, and the portion spotted with the antibody solution is colored. The colored image can be visually confirmed to confirm the presence or absence of a physiologically active substance to be detected. In addition, the colored image can be captured using an image scanner or a CCD camera, and the degree of color development can be digitized by image processing software (for example, NIH image) to compare the amount of the physiologically active substance to be detected.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
(Example)
Saturated cyclic polyolefin resin (hydrogenated ring-opening polymer of 5-methyl-2-norbornene, MFR (Melt flow rate): 21 g / 10 min, hydrogenation rate: substantially 100%, heat distortion temperature 123 ° C.) A solid-phase substrate was prepared by processing into a slide glass shape (size: 76 mm × 26 mm × 1 mm). This solid phase substrate was made of polyethylene glycol methyl ether methacrylate (also known as methoxypolyethylene glycol methacrylate) -p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate-3-methacryloxypropyldimethylethoxysilane (PEGMA-MEONP-MPDES, each group in mol%). 92: 3: 5) is immersed in a 0.3% by weight ethanol solution and dried by heating at 65 ° C. for 4 hours to have an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue and an active ester group on the substrate surface. A substrate having a layer containing a polymer material copolymerized with an ethylenically unsaturated polymerizable monomer and an ethylenically unsaturated polymerizable monomer having a crosslinkable functional group was obtained.

(Comparative example)
Saturated cyclic polyolefin resin (hydrogenated ring-opening polymer of 5-methyl-2-norbornene, MFR (Melt flow rate): 21 g / 10 min, hydrogenation rate: substantially 100%, heat distortion temperature 123 ° C.) A solid-phase substrate was prepared by processing into a slide glass shape (size: 76 mm × 26 mm × 1 mm). This solid phase substrate was subjected to a hydrophilic treatment on the surface by low-temperature oxygen plasma treatment. Next, a solution in which γ-aminopropyltriethoxysilane is dissolved as an aminoalkylsilane in methanol at a concentration of 5% by weight is prepared as an amino group introduction treatment solution, immersed in this solution for 2 hours, and then the substrate. Was taken out of the solution, immersed in ultrapure water and allowed to stand, and then the substrate was taken out and dried. Glutaraldehyde was dissolved in PBS (-) at a concentration of 2% by weight to prepare a glutaraldehyde solution. The substrate treated with aminoalkylsilane was immersed in the glutaraldehyde solution and allowed to stand for 4 hours. It was taken out, immersed in ultrapure water, washed and dried. As a result, an aldehyde substrate having an aldehyde group on the surface was obtained.

(Immobilization and blocking of primary antibody)
Next, a rat-derived anti-mouse IgG2a antibody solution prepared to a concentration of 0.5 mg / mL with a carbonate buffer of pH 9.5 was spotted on the substrates obtained in Examples and Comparative Examples using a micropipette. This substrate was allowed to stand at room temperature for 24 hours to be fixed. Thereafter, each substrate was subjected to blocking treatment and washed with PBS containing 0.05% Tween20.

(Reaction between immobilized antibody and antigen)
Next, a mouse IgG2a solution is developed on the substrate surface at each antigen concentration shown in Table 1, and left to stand at a humidity of 99% and 37 ° C. for 1 hour to cause an antigen-antibody reaction, and contains 0.05% Tween 20 Washed with PBS.

(Reaction between antigen and secondary antibody)
Next, a biotin-labeled anti-mouse IgG2a antibody solution prepared at a concentration of 0.5 μg / mL is developed on the surface of the substrate and allowed to stand for 1 hour at 37 ° C. in a humidity of 99% to cause an antigen-antibody reaction. Washing was performed with PBS containing 05% Tween20.

(Biotin-avidin reaction)
Next, an alkaline phosphatase solution labeled with streptavidin is developed on the surface of the substrate, left to stand at a humidity of 99% and 37 ° C. for 30 minutes to cause a reaction, and washed with PBS containing 0.05% Tween20. It was.

(Coloring reaction)
Subsequently, the substrate was immersed in a BCIP / NBT solution, allowed to stand at 37 ° C. for 30 minutes, and then washed with pure water. In the comparative example, the coloring of the spotted portion of the antibody was hardly observable with the naked eye, but in the example, the coloring of the spotted portion was clearly observable. A colored image of the spotted portion of the antibody on the substrate was captured by a CCD camera, and the captured digital data was processed by image processing software (NIH image) to quantify the degree of coloring. The results are shown in Table 1.

  In the examples, the degree of coloring depending on the antigen concentration was seen, and the antigen could be detected by the antigen-antibody reaction on the substrate, but in the comparative example, the efficiency of the antigen-antibody reaction was poor, and the efficiency of the enzyme reaction was poor, The coloring reaction efficiency of the chromogenic substrate was poor, and the antigen could not be detected. That is, in the method for detecting a physiologically active substance of the present invention, the physiologically active substance can be detected in the visible region without using a fluorescent dye, without using a special reading device, and the presence or absence of the physiologically active substance. It can be said that it was possible to quantify not only the judgment.

Claims (21)

Ethylene unsaturated polymerizable monomer (A) having an alkylene glycol residue, ethylene unsaturated polymerizable monomer (B) having a functional group for immobilizing a physiologically active substance, and ethylene unsaturated having a crosslinkable functional group Using an insoluble carrier having a polymer substance copolymerized with the polymerizable monomer (C) on the surface,
(A) producing an antibody-immobilized carrier by immobilizing an antibody on the surface of the insoluble carrier;
(B) adding a solution containing a physiologically active substance as an antigen on the antibody-immobilized carrier and reacting with the antibody;
(C) binding an enzyme-containing substance to the reacted antibody-antigen complex;
(D) adding a solution containing a chromogenic substrate and causing the chromogenic substrate to develop color by the reaction of the enzyme;
And detecting the content of the physiologically active substance in the solution in step (b) according to the degree of color development. In addition to the above steps,
(B-2) a step of further adding a solution containing a secondary antibody to the reacted antibody-antigen complex and reacting with the complex;
The method for detecting a physiologically active substance according to claim 1, comprising: In step (b-2), the secondary antibody is preliminarily labeled with biotin, and the solution containing the enzyme-labeled avidin in step (c) is brought into contact with the carrier surface to label the enzyme on the antibody-antigen complex. The method for detecting a physiologically active substance according to claim 2. The method for detecting a physiologically active substance according to any one of claims 1 to 3, wherein the enzyme labeled with the antibody-antigen complex is an oxidation or reductase. The method for detecting a physiologically active substance according to any one of claims 1 to 4, wherein the enzyme to be labeled is peroxidase or alkaline phosphatase. The method for detecting a physiologically active substance according to any one of claims 1 to 5, wherein in the step (a), the antibody is immobilized by covalent bonding to the surface of the carrier. The method for detecting a physiologically active substance according to any one of claims 1 to 6, wherein the ethylenically unsaturated polymerizable monomer (A) having an alkylene glycol residue is a monomer represented by the following general formula [1].

(Wherein R ₁ represents a hydrogen atom or a methyl group, R ₄ represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms, X represents an alkylene glycol residue having 1 to 10 carbon atoms, and p represents 1 to Represents an integer of 100. The repeated Xs may be the same or a chain of different alkylene glycol groups. The method for detecting a physiologically active substance according to claim 7, wherein the ethylenically unsaturated polymerizable monomer (A) having an alkylene glycol residue is methoxypolyethylene glycol (meth) acrylate. The method for detecting a physiologically active substance according to claim 8, wherein the average chain of ethylene glycol in the methoxypolyethylene glycol (meth) acrylate is 3 to 100. The functional group of the ethylenically unsaturated polymerizable monomer (B) having a functional group for immobilizing a physiologically active substance is at least one functional group selected from an aldehyde group, an active ester, an epoxy group, a vinyl sulfone group, and biotin. Item 10. A method for detecting a physiologically active substance according to any one of Items 1 to 9. The physiology according to any one of claims 1 to 9, wherein the ethylenically unsaturated polymerizable monomer (B) having a functional group for immobilizing a physiologically active substance is a monomer having an active ester represented by the following general formula [2]. Active substance detection method.

(In the formula, R ₂ represents a hydrogen atom or a methyl group, Y represents an alkylene glycol residue or an alkyl group having 1 to 10 carbon atoms, W represents an active ester group, and q represents an integer of 1 to 20.) When q is an integer of 2 or more and 20 or less, each repeated Y may be the same or a chain of different alkylene glycol residues. The method for detecting a physiologically active substance according to claim 10 or 11, wherein the active ester is p-nitrophenyl ester or N-hydroxysuccinimide ester. 13. The crosslinkable functional group of the ethylenically unsaturated polymerizable monomer (C) having a crosslinkable functional group is at least one functional group selected from alkoxysilyl, epoxy, and (meth) acryl. Or a method for detecting a physiologically active substance. The detection of a physiologically active substance according to any one of claims 1 to 12, wherein the ethylenically unsaturated polymerizable monomer (C) having a crosslinkable functional group is a monomer having alkoxysilyl represented by the following general formula [3]. Method.

(In the formula, R ₃ represents a hydrogen atom or a methyl group, and Z represents an alkyl group having 1 to 20 carbon atoms. At least one of A ₁ , A ₂ , and A ₃ is a hydrolyzable alkoxy group. And others represent alkyl groups.) The method for detecting a physiologically active substance according to any one of claims 1 to 14, wherein the colored dye adheres to the surface of the antibody or antigen and a nearby carrier, and the degree of adhesion of the dye is measured as the color intensity. . The method for detecting a physiologically active substance according to claim 1, wherein the degree of color development is measured by absorption. The method for detecting a physiologically active substance according to any one of claims 1 to 16, wherein the antibody is immobilized in a spot shape on the surface of a carrier. The method for detecting a physiologically active substance according to claim 17, wherein spots of a plurality of types of antibodies are present in the same compartment on the surface of the carrier. The shape of the carrier is at least one selected from the group consisting of a glass slide, a 96-hole plate, a 384-hole plate, a 1536-hole plate, a microchannel, a bead, a tube, a container, and a complex thereof. Item 19. A method for detecting a physiologically active substance according to any one of Items 1 to 18. The method for detecting a physiologically active substance according to any one of claims 1 to 19, wherein the carrier is made of plastic. 21. The method for detecting a physiologically active substance according to claim 20, wherein the plastic is at least one selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefin, polypentene, polyamide, and copolymers thereof.