JP4228101B2 - Antibody immobilization method and antibody immobilization substrate - Google Patents
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本発明は、イムノアッセイや抗原の分画、精製などに使用可能な抗体を固定する方法およびその方法を用いて形成した抗体固定基板に関するものである。 The present invention relates to a method for immobilizing an antibody that can be used for immunoassay, antigen fractionation, purification, and the like, and an antibody immobilization substrate formed using the method.
従来より良く知られているように抗体は抗原と特異的に結合する性質があり、イムノアッセイや抗原物質の分画、精製などに使用されている。その際、抗体はプラスチック、金属などの不溶性物質に固定した形で使用される。なお、これら不溶性物質の多くは生体高分子を非特異的に吸着する性質を持つ。 As is well known in the art, an antibody has a property of specifically binding to an antigen and is used for immunoassay, fractionation and purification of an antigenic substance. In that case, the antibody is used in a form immobilized on an insoluble material such as plastic or metal. Many of these insoluble substances have the property of non-specifically adsorbing biopolymers.
抗体の不溶性物質への固定化方法の一つとして、不溶性物質上の抗体結合分子を介して抗体を不溶性物質に固定化する方法がある。この場合、抗体結合分子は共有結合または静電相互作用などの非共有結合を介して不溶性物質に固定化される（例えば、特許文献１参照）。 One method for immobilizing antibodies to insoluble substances is to immobilize antibodies to insoluble substances via antibody binding molecules on the insoluble substances. In this case, the antibody-binding molecule is immobilized on an insoluble substance via a non-covalent bond such as a covalent bond or electrostatic interaction (see, for example, Patent Document 1).
前記従来の方法では次のような課題がある。
（１）不溶性物質が生体高分子を非特異的に吸着するため、それらがイムノアッセイや分画、精製の妨げになる可能性がある。
（２）不溶性物質と抗体結合分子が結合を形成するため両者が極めて近い位置に存在することになり、その結果引き起こされる２次的（非特異的）な不溶性物質−抗体結合分子間の相互作用により、抗体結合分子の抗体結合活性が失われる可能性がある。
The conventional method has the following problems.
(1) Since insoluble substances adsorb biopolymers nonspecifically, they may interfere with immunoassay, fractionation, and purification.
(2) Since the insoluble substance and the antibody-binding molecule form a bond, they are located at very close positions. As a result, the interaction between the secondary (non-specific) insoluble substance and the antibody-binding molecule is caused. As a result, the antibody binding activity of the antibody binding molecule may be lost.
本発明の目的は、上記の課題を解決するもので、アミド基を持つゲルを用いて抗体結合分子の非特異的な吸着を抑えると共に、抗体結合分子を前記アミド基を持つゲル内に包接固定して抗体結合分子の抗体結合活性低下を抑制して、抗体分子をアミド基を持つゲルあるいは不溶性物質に敷設されたアミド基を持つゲルに固定することのできる抗体固定方法および抗体固定基板を提供することにある。 An object of the present invention is to solve the above-mentioned problems, and suppress nonspecific adsorption of antibody-binding molecules by using a gel having an amide group, and include the antibody-binding molecule in the gel having the amide group. An antibody immobilization method and an antibody immobilization substrate capable of immobilizing and immobilizing an antibody molecule on a gel having an amide group or a gel having an amide group laid on an insoluble substance by suppressing the decrease in antibody binding activity of the antibody-binding molecule It is to provide.
このような課題を達成するために、本発明の請求項１に記載の抗体固定方法では、
不溶性物質で形成された基板上に、抗体結合分子を非共有結合的に包接したアミド基を持つゲルを２次元または３次元状に固定し、前記抗体結合分子に抗体分子の根元部を固定することを特徴とする。
In order to achieve such a problem, in the antibody immobilization method according to claim 1 of the present invention,
On a substrate formed of an insoluble substance, a gel having an amide group that non-covalently encapsulates an antibody-binding molecule is fixed two-dimensionally or three-dimensionally, and the root of the antibody molecule is fixed to the antibody-binding molecule. It is characterized by doing.
このように、アミド基を持つゲルに包接された抗体結合分子を介して抗体分子の根元部を基板に固定すると、次のような効果がある。
（１）アミド基を持つ分子はタンパク質を吸着しにくいため、非特異的吸着による抗原−抗体反応の阻害、アッセイ時の測定誤差または精製時の純度低下を引き起こす不純物の混入を避けることができる。
（２）アミノ基を持つゲルにより抗体結合分子を包接するため、担体との結合形成に起因する抗体結合活性の低下を抑制することができる。
（３）抗体分子の根元部が抗体結合分子に固定されるため、抗体の抗原と結合する抗原認識部位が基板面から上向きにフリーとなって抗体の配向性が向上し、抗原との特異的結合性が増大する。
As described above, fixing the base portion of the antibody molecule to the substrate through the antibody binding molecule included in the gel having the amide group has the following effects.
(1) Since a molecule having an amide group is difficult to adsorb proteins, it is possible to avoid contamination of impurities that cause inhibition of antigen-antibody reaction due to non-specific adsorption, measurement error during assay, or purity reduction during purification.
(2) Since the antibody binding molecule is included by the gel having an amino group, it is possible to suppress a decrease in the antibody binding activity due to the bond formation with the carrier.
(3) Since the base of the antibody molecule is fixed to the antibody binding molecule, the antigen recognition site that binds to the antigen of the antibody is free upward from the substrate surface, improving the orientation of the antibody, and specific to the antigen Connectivity is increased.
この場合、抗体結合分子としては、請求項２のように、プロテインＧを代表とする抗体結合活性を持つ生体高分子または高分子化合物が使用できる。
また、不溶性物質としては、請求項３のように、金属、プラスチック、ガラスなどの不溶性高分子化合物が使用できる。
In this case, as the antibody binding molecule, a biopolymer or a polymer compound having antibody binding activity represented by protein G can be used as in
Further, as the insoluble substance, an insoluble polymer compound such as metal, plastic and glass can be used as in the third aspect.
また、アミド基を持つゲルとしては、例えば、請求項４に記載のように、ポリアクリルアミドゲルが用いられる。
また、抗体結合分子と抗体分子は、請求項５のように、１点または多点で結合する。
また、アミド基を持つゲルは、請求項６のように、メタクリロキシプロピルトリメトキシシランで処理された不溶性物質の表面に塗布・重合される。
As the gel having an amide group, for example, a polyacrylamide gel is used as described in
Further, the antibody-binding molecule and the antibody molecule are bonded at one point or multiple points as in the fifth aspect.
The gel having an amide group is coated and polymerized on the surface of an insoluble material treated with methacryloxypropyltrimethoxysilane as described in claim 6.
本発明の請求項７に記載の抗体固定基板では、
抗体と結合する抗体結合分子を非共有結合的に包接するアミド基を持つゲルを、不溶性物質に敷設してなることを特徴とする。
このような構成の基板によれば、請求項１の抗体固定方法における場合と同様に、非特異的吸着による抗原−抗体反応の阻害、アッセイ時の測定誤差または精製時の純度低下を引き起こす不純物の混入を避けることができ、担体との結合形成に起因する抗体結合活性の低下を抑制することができ、抗体の配向性が向上して抗原との特異的結合性が増大するなどの効果がある。
In the antibody-immobilized substrate according to claim 7 of the present invention,
A gel having an amide group that non-covalently encloses an antibody-binding molecule that binds to an antibody is laid on an insoluble substance.
According to the substrate having such a structure, as in the antibody immobilization method of claim 1, impurities that cause inhibition of antigen-antibody reaction by non-specific adsorption, measurement error during assay, or purity reduction during purification can be obtained. It is possible to avoid contamination, suppress the decrease in antibody binding activity due to the formation of a bond with the carrier, and improve the orientation of the antibody and increase the specific binding to the antigen. .
この場合、抗体結合分子としては、プロテインＧを代表とする抗体結合活性を持つ生体高分子または高分子化合物であり、その抗体結合分子は前記アミド基に非共有結合的に包接されており（請求項８）、不溶性物質としては金属、プラスチック、ガラスなどの不溶性材料が使用でき（請求項９）、アミド基を持つゲルにはポリアクリルアミドゲルが用いられ（請求項１０）、アミド基を持つゲルは不溶性物質の表面をメタクリロキシプロピルトリメトキシシランで処理した上に塗布し重合したものとすることができる（請求項１１）。 In this case, the antibody binding molecule is a biopolymer or a polymer compound having antibody binding activity typified by protein G, and the antibody binding molecule is non-covalently included in the amide group ( (Claim 8) Insoluble materials such as metals, plastics and glass can be used as insoluble substances (Claim 9), and polyacrylamide gels are used as gels having amide groups (Claim 10). The gel can be obtained by polymerizing a surface of an insoluble substance treated with methacryloxypropyltrimethoxysilane (claim 11).
以上説明したことから明らかなように、本発明によれば次のような効果がある。
（１）アミド基を持つゲルに抗体結合分子を包接し、その抗体結合分子に抗体分子の根元部を固定するため、非特異的吸着が少なく、しかも抗体の配向性が向上して抗原との特異的結合性も増大する抗体固定方法および基板を容易に実現することができる。
（２）抗体結合分子を包接固定することで、担体との結合形成に起因する抗体結合活性の低下を抑制できる。
（３）上記（１）、（２）項により、高感度のイムノアッセイや抗原の分画、精製などに使用可能な抗体固定基板の調製が可能となる。
As is apparent from the above description, the present invention has the following effects.
(1) Since an antibody binding molecule is included in a gel having an amide group, and the root of the antibody molecule is fixed to the antibody binding molecule, non-specific adsorption is reduced, and the orientation of the antibody is improved. Antibody immobilization methods and substrates that also increase specific binding can be easily realized.
(2) By including and fixing antibody-binding molecules, it is possible to suppress a decrease in antibody-binding activity due to bond formation with a carrier.
(3) The above items (1) and (2) make it possible to prepare an antibody-immobilized substrate that can be used for highly sensitive immunoassay, antigen fractionation, purification, and the like.
本発明では、基板材料としてポリアクリルアミドゲルを用い、さらに抗体結合分子がポリアクリルアミドゲルに包接されるように基板を調製することで、担体との結合形成による抗体結合分子の活性低下が起きない状態を作り出している。 In the present invention, a polyacrylamide gel is used as the substrate material, and the substrate is prepared so that the antibody-binding molecule is included in the polyacrylamide gel, so that the activity of the antibody-binding molecule does not decrease due to bond formation with the carrier. Creating a state.
また、本発明においては、抗体結合分子をポリアクリルアミドゲルにより包接するため、担体との結合形成に起因する抗体結合活性の低下が起きない。また、抗体結合分子が活性を持つ溶液状態に比較的近い状態で包接物質内に保持されることが期待される。なお、包接による固定化は抗体結合分子内の官能基に依存せず、包接物質が形成する孔のサイズ主に依存する。 In the present invention, since antibody binding molecules are included by polyacrylamide gel, the antibody binding activity is not reduced due to the bond formation with the carrier. In addition, it is expected that the antibody-binding molecule is retained in the inclusion substance in a state relatively close to a solution state in which the antibody binding molecule is active. Immobilization by inclusion does not depend on the functional group in the antibody binding molecule, but mainly depends on the size of the pore formed by the inclusion substance.
このようにして調製した抗原結合分子を含むポリアクリルアミドゲルはそのまま、もしくは不溶性物質に敷設した形で抗体固定基板として供される。抗体固定化はポリアクリルアミドゲルまたはポリアクリルアミドゲルを敷設した不溶性物質を、抗体溶液中に浸漬することで行う。 The polyacrylamide gel containing the antigen-binding molecule thus prepared is used as an antibody immobilization substrate as it is or in a form laid on an insoluble substance. Antibody immobilization is performed by immersing a polyacrylamide gel or an insoluble substance laid with a polyacrylamide gel in an antibody solution.
以下図面を用いて本発明を詳しく説明する。図１は本発明に係る抗体分子結合方法の原理を説明する図、図２はポリアクリルアミドゲル基板上への抗体固定の態様を示す模式図である。 Hereinafter, the present invention will be described in detail with reference to the drawings. FIG. 1 is a diagram for explaining the principle of an antibody molecule binding method according to the present invention, and FIG. 2 is a schematic diagram showing an embodiment of antibody immobilization on a polyacrylamide gel substrate.
抗体分子の結合手順を以下に説明する。図１（ａ）に示すようにアミド基１と抗体結合分子２を混合し、同図（ｂ）に示すように不溶性物質３上に塗布後、重合反応を行わせる。不溶性物質３としては金属、プラスチック、ガラスなどの不溶性材料が使用される。この重合反応により、抗体結合分子２はアミド基を持つゲル（以下ポリアクリルアミドゲルを例にとって説明する）が形成する網目構造に包み込まれる形になる（同図（ｃ））。
The procedure for binding antibody molecules is described below. As shown in FIG. 1 (a), amide group 1 and antibody-binding
抗体を固定する際には、同図（ｄ）に示すように抗体分子４を含む溶液を滴下する。抗体分子としてはモノクローナル抗体またはポリクローナル抗体が使用される。抗体分子４は自身の抗体分子結合部位５を介して、ポリアクリルアミドゲルに包接固定された抗体結合分子２と１点または多点で結合する。なお、抗体結合分子２は非共有結合的にポリアクリルアミドゲルに包接されるよう調整される。これにより、抗体分子４は、その根元部が抗体結合分子２に固定され、抗原と結合する部位が上向きにフリーの状態となる。
When the antibody is immobilized, a solution containing the
図２は、このような原理に基づいて抗体分子を固定化することのできる抗体固定基板の説明図である。図では、ポリアクリルアミドゲル１０に包接された抗体結合分子２により抗体分子４が固定された（異なる種類の抗体Ａ、抗体Ｂなどでアドレッシングされた）様子も示してある。
FIG. 2 is an explanatory diagram of an antibody immobilization substrate on which antibody molecules can be immobilized based on such a principle. The figure also shows a state in which the
図において、２０は不溶性物質で、例えばガラス基板である。ガラス基板２０の表面をメタクリロキシプロピルトリメトキシシラン３０で処理し、その上にポリアクリルアミドゲル１０を塗布して重合させる。これにより、ポリアクリルアミドゲル１０をガラス基板２０に強く固定することができる。なお、ポリアクリルアミドゲル１０はガラス基板２０上に２次元または３次元状に敷設固定することができる。
In the figure, 20 is an insoluble substance, for example, a glass substrate. The surface of the glass substrate 20 is treated with
ポリアクリルアミドゲル１０には、図１に示すように抗体結合分子（例えばプロテインＧ）２が非共有結合的に包接固定され、抗体分子４は図２に示すようにその根元部が抗体結合分子２に固定され、その抗原認識部位が溶液方向（図では上方）に向くように固定される。このように固定された抗体分子４の抗原認識部位には対応の抗原が結合する。すなわち、抗体Ａの抗体分子の抗原認識部位には抗体Ａの抗原が、抗体Ｂの抗原認識部位には抗体Ｂの抗原がそれぞれ結合する。
As shown in FIG. 1, an antibody-binding molecule (for example, protein G) 2 is included and immobilized on polyacrylamide gel 10 in a non-covalent manner, and
このように、ポリアクリルアミドゲル１０中に埋め込んだ抗体結合分子２を介して抗体分子を基板に固定すると、非特異的吸着は極めて少なく、抗体の抗原認識部位４ａが溶液方向に向いて配向性が向上し、抗体抗原の結合効率が一段と向上する。
As described above, when the antibody molecule is immobilized on the substrate through the
以上の説明は、本発明の説明および例示を目的として特定の好適な実施例を示したに過ぎない。したがって本発明は、上記実施例に限定されることなく、その本質から逸脱しない範囲で更に多くの変更、変形をも含むものである。 The foregoing description is merely illustrative of certain preferred embodiments for purposes of explanation and illustration of the invention. Therefore, the present invention is not limited to the above-described embodiments, and includes many changes and modifications without departing from the essence thereof.
１ アクリルアミド
２ 抗体結合分子
３ 不溶性物質
４ 抗体分子
５ 抗体分子結合部位
１０ ポリアクリルアミドゲル
２０ ガラス基板
３０ メタクリロキシプロピルトリメトキシシラン
DESCRIPTION OF SYMBOLS 1
Claims (11)
前記抗体と結合する抗体結合分子を非共有結合的に包接するアミド基を持つゲルを、不溶性物質に敷設してなる抗体固定基板。 A substrate on which an antibody is immobilized,
An antibody immobilization substrate comprising a gel having an amide group that non-covalently encloses an antibody-binding molecule that binds to the antibody, laid on an insoluble substance.
The antibody-immobilized substrate according to any one of claims 7 to 10, wherein the gel having the amide group is applied and polymerized after the surface of the insoluble substance is treated with methacryloxypropyltrimethoxysilane. .
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| JP2003417494A JP4228101B2 (en) | 2003-12-16 | 2003-12-16 | Antibody immobilization method and antibody immobilization substrate |
| DE102004048685A DE102004048685A1 (en) | 2003-10-06 | 2004-10-06 | Process to demonstrate the presence of a bipolymer in a sample by trapping a target bipolymer on a substrate |
| US10/960,849 US20050191644A1 (en) | 2003-10-06 | 2004-10-06 | Methods for detecting biopolymers; biochips; methods for immobilizing antibodies; and substrates to which antibodies are immobilized |
| CN200410090010.5A CN1629636A (en) | 2003-10-06 | 2004-10-08 | Method of detecting biopolymer, biochip, method of fixing antibody, and substrate for fixing antibody thereon |
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