JPH05273212A - Method for immobilizing antibody protein by protein a molecular film and antibody immobilized film - Google Patents
Method for immobilizing antibody protein by protein a molecular film and antibody immobilized filmInfo
- Publication number
- JPH05273212A JPH05273212A JP4003257A JP325792A JPH05273212A JP H05273212 A JPH05273212 A JP H05273212A JP 4003257 A JP4003257 A JP 4003257A JP 325792 A JP325792 A JP 325792A JP H05273212 A JPH05273212 A JP H05273212A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- protein
- film
- immobilized
- membrane
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 62
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims description 27
- 230000003100 immobilizing effect Effects 0.000 title claims description 7
- 239000002120 nanofilm Substances 0.000 title 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000003018 immunoassay Methods 0.000 claims abstract description 8
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- 239000012528 membrane Substances 0.000 claims description 48
- 239000007787 solid Substances 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 239000000427 antigen Substances 0.000 abstract description 23
- 102000036639 antigens Human genes 0.000 abstract description 23
- 108091007433 antigens Proteins 0.000 abstract description 23
- 239000000758 substrate Substances 0.000 abstract description 21
- 108010071390 Serum Albumin Proteins 0.000 abstract description 4
- 102000007562 Serum Albumin Human genes 0.000 abstract description 4
- 239000011521 glass Substances 0.000 abstract description 4
- 230000009257 reactivity Effects 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 10
- 229940027941 immunoglobulin g Drugs 0.000 description 9
- 239000002356 single layer Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 239000008055 phosphate buffer solution Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 108091006905 Human Serum Albumin Proteins 0.000 description 3
- 102000008100 Human Serum Albumin Human genes 0.000 description 3
- 230000001186 cumulative effect Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- PYJJCSYBSYXGQQ-UHFFFAOYSA-N trichloro(octadecyl)silane Chemical compound CCCCCCCCCCCCCCCCCC[Si](Cl)(Cl)Cl PYJJCSYBSYXGQQ-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000005501 phase interface Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は抗体タンパクの固定化法
に関する。更に詳しくは、バイオセンサー、バイオリア
クター、バイオエレクトロニクスデバイス、免疫測定基
盤に有用な抗体固定膜を得るために、ラングミュア・ブ
ロジェット（ＬＢ）法を利用して、抗原抗体反応の活性
を保持した状態で抗体タンパクを固体基盤上に高密度に
固定する方法、その方法により得られた抗体固定膜及び
該抗体固定膜を用いたバイオセンサー等に関する。FIELD OF THE INVENTION The present invention relates to a method for immobilizing antibody proteins. More specifically, in order to obtain an antibody-immobilized membrane useful for biosensors, bioreactors, bioelectronic devices, and immunoassay platforms, the Langmuir-Blodgett (LB) method is used to maintain the activity of the antigen-antibody reaction. And a method for immobilizing antibody proteins on a solid substrate with high density, an antibody-immobilized membrane obtained by the method, a biosensor using the antibody-immobilized membrane, and the like.
【０００２】[0002]
【従来の技術】いわゆる抗体固定化法には、（１）抗体
タンパクのアミノ基またはカルボキシル基と、反応また
は吸着結合出来る官能基を有する固体表面上に固定化す
る方法、（２）親水ゲル中に抗体タンパクを抱き込ませ
て、固体基盤上に固定化する方法、及び（３）ＬＢ法を
利用した抗体固定化法として、水面に脂質膜を展開し、
水層中から抗体タンパクを吸着又は取り込ませる方法
（J. Cell. Biochem.，29 239(1985)．)或いは水面に水
不溶性ポリ（オレフィン−無水マレイン酸）単分子膜に
当該水相中に溶解した水溶性抗体タンパク質を接触させ
ることにより当該水相界面で抗体タンパク−単分子膜複
合体を形成させ、それを固体基板上に積層する方法（特
開昭６３−３８１６４号）が知られている。2. Description of the Related Art The so-called antibody immobilization method includes (1) immobilization on a solid surface having a functional group capable of reacting or adsorbing with an amino group or a carboxyl group of an antibody protein, (2) in hydrophilic gel As an antibody immobilization method using the LB method by immobilizing the antibody protein on the solid substrate by hugging the antibody protein on it, a lipid membrane is developed on the water surface,
A method of adsorbing or incorporating an antibody protein from the water layer (J. Cell. Biochem., 29 239 (1985).) Or dissolving in a water-insoluble poly (olefin-maleic anhydride) monomolecular film in the water phase. A method is known in which an antibody protein-monolayer complex is formed at the aqueous phase interface by contacting the water-soluble antibody protein described above, and is laminated on a solid substrate (JP-A-63-38164). ..
【０００３】[0003]
【発明が解決しようとする課題】前記(1)の方法では化
学反応により固体表面に結合させるため、固定化の反応
条件によっては、抗体タンパクの変性や、非特異的反応
による抗原認識部位の変性が起こり易く、前記（２）の
方法では抗原がゲル中の抗体と接触しにくいため、抗原
抗体反応が阻止され易い等の問題を有していた。また
（３）のＬＢ法を利用した抗体固定化法では抗体と脂質
膜の結合は吸着結合であるため、抗体が離脱し易いとい
う問題を有しており、また、従来のこの種の方法では抗
体タンパクをＬＢ膜作成用の水層に溶解して固定化する
ため、貴重な抗体を多量に必要とするという欠点があっ
た。そして従来の抗体固定膜を用いた免疫測定法では、
（１）抗原、抗体の固相化に１夜、（２）測定物との反
応に数時間、（３）２次抗体の反応、（４）酵素反応、
等測定のためのステップ数が多く、測定終了までに長い
時間を要していた。又、バイオセンサー、バイオリアク
ター、バイオエレクトロニクスデバイス、免疫測定基盤
の作成には、抗体固定膜は高い反応性、高感度、即ち固
定化抗体量が多いこと、速い応答性、微小化、即ち超薄
膜であることが要望されている。しかし、従来のもので
は抗体密度が低いこと、膜が厚いことからこれらバイオ
センサー等の作成は困難であった。In the above method (1), since it is bound to a solid surface by a chemical reaction, denaturation of the antibody protein or denaturation of the antigen recognition site by a non-specific reaction may occur depending on the reaction conditions for immobilization. And the method (2) has a problem that the antigen-antibody reaction is likely to be blocked because the antigen is less likely to come into contact with the antibody in the gel. Further, in the antibody immobilization method using the LB method of (3), since the binding between the antibody and the lipid membrane is an adsorption bond, there is a problem that the antibody is easily released, and in the conventional method of this kind, Since the antibody protein is dissolved and immobilized in the aqueous layer for forming the LB film, there is a drawback that a large amount of valuable antibody is required. And in the conventional immunoassay using the antibody-immobilized membrane,
(1) overnight for immobilization of antigen and antibody, (2) several hours for reaction with analyte, (3) reaction of secondary antibody, (4) enzyme reaction,
There are many steps for equal measurement, and it took a long time to complete the measurement. In addition, for the preparation of biosensors, bioreactors, bioelectronic devices, and immunoassay substrates, antibody-immobilized membranes have high reactivity and high sensitivity, that is, the amount of immobilized antibody is large, fast response, miniaturization, that is, ultrathin film. Is required. However, it was difficult to produce these biosensors and the like due to the low antibody density and the thick film in the conventional ones.
【０００４】[0004]
【課題を解決するための手段】本発明者らは、上記の問
題を解決すべく鋭意研究の結果、単分子膜形成物質とし
てプロテインＡを用い、プロテインＡから得られた膜に
抗体を固定化したとき、従来の抗体固定化方法の問題を
解決し得、それにより得られた抗体固定化膜は従来の抗
体固定化膜に比して、抗体が離脱することなく、抗体活
性、抗体密度が高く、かつ均一な抗体固定膜であること
を見出し、本発明に到達したのである。即ち、本発明
は、（１）プロテインＡの水溶液を水面に滴下展開し、
形成された膜を膜が破壊されるより低い表面圧力で圧縮
保持し、固体表面に移し取った後、プロテインＡの膜上
に抗体タンパク質を作用させることにより、抗体タンパ
クを固定化する方法、（２）該方法により得られた抗体
固定膜、（３）該抗体固定膜を用いたバイオセンサー、
バイオリアクターもしくはバイオエレクトロニクスデバ
イス及び（４）該抗体固定膜を用いた酵素測定法に関す
る。本発明では、水層表面に展開されたプロテインＡを
ＬＢ法により固体基盤に写し取り、プロテインＡの単分
子膜、あるいは累積膜上に抗体タンパクの溶液を接触さ
せ、プロテインＡ・ＬＢ膜上に抗体タンパク質を固定化
するものであり、上記の方法で得られた抗体固定膜を用
いたバイオセンサー、バイオリアクター、バイオエレク
トロニクスデバイス、免疫測定基盤に関する。本発明で
用いるプロテインＡは黄色ブドウ球菌の菌体表面、ある
いは菌体外に放出される、分子量が１５０００から５２
０００のタンパク質である。又、本発明でいう抗体タン
パク質とは、免疫グロブリンＧ（ＩｇＧ）である。Ｉｇ
Ｇは疎水性末端部位（Ｆｃ）と抗原と特異的に反応する
抗原認識部位（Ｆａｂ）を持つ分子量約１５００００の
タンパク質である。プロテインＡは哺乳動物の免疫グロ
ブリン、特にＩｇＧのサブクラスであるＩｇＧ1、Ｉｇ
Ｇ2、ＩｇＧ4、のＦｃ部位と特異的に結合する性質があ
り、この性質を利用して高い抗体密度の抗体固定化が実
現できる。Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have used protein A as a monomolecular film-forming substance and immobilized an antibody on a film obtained from protein A. Then, the problem of the conventional antibody immobilization method can be solved, and the antibody-immobilized membrane thus obtained has a higher antibody activity and a higher antibody density than the conventional antibody-immobilized membrane without releasing the antibody. The inventors have found that the membrane is high and uniform, and arrived at the present invention. That is, the present invention is: (1) An aqueous solution of protein A is dropped and developed on the water surface,
A method for immobilizing an antibody protein by allowing the formed membrane to be compressed and held at a lower surface pressure than that at which the membrane is broken, transferred to a solid surface, and then causing the antibody protein to act on the protein A membrane, ( 2) an antibody-immobilized membrane obtained by the method, (3) a biosensor using the antibody-immobilized membrane,
The present invention relates to a bioreactor or a bioelectronic device and (4) an enzyme measuring method using the antibody-immobilized membrane. In the present invention, the protein A developed on the surface of the aqueous layer is transferred onto a solid substrate by the LB method, and a solution of the antibody protein is brought into contact with the monomolecular film of the protein A or the cumulative film to form the protein A / LB film. The present invention relates to a biosensor, a bioreactor, a bioelectronic device, and an immunoassay platform for immobilizing an antibody protein and using the antibody-immobilized membrane obtained by the above method. The protein A used in the present invention has a molecular weight of 15,000 to 52, which is released on the surface of Staphylococcus aureus or outside the cells.
000 proteins. The antibody protein referred to in the present invention is immunoglobulin G (IgG). Ig
G is a protein having a molecular weight of about 150,000 and having a hydrophobic terminal region (Fc) and an antigen recognition site (Fab) that specifically reacts with an antigen. Protein A is a mammalian immunoglobulin, especially IgG 1 , Ig which is a subclass of IgG.
It has a property of specifically binding to the Fc sites of G 2 and IgG 4 , and by utilizing this property, antibody immobilization with high antibody density can be realized.
【０００５】本発明の抗体固定化膜は例えば以下のよう
な方法でＬＢ法を適用して得ることが出来る。即ち、プ
ロテインＡを溶媒に溶解し、この溶液をＬＢ装置におい
て水面上に滴下、あるいは流下し展開させる。気液界面
にはプロテインＡ単分子膜が形成される。この膜を膜が
破壊される圧力より低い表面圧に圧縮保持し、固体基盤
上に写し取る。単分子膜の累積を行う場合は、なるべく
高い表面圧力に圧縮することが好ましい。ここで溶媒は
水あるいはリン酸等の緩衝溶液であり、プロテインＡの
濃度は０．０５から１ｇ／ｌである。又、膜への圧縮圧
はプロテインＡが単分子状態を保つことが出来る圧力で
通常７から１３ｍＮ／ｍである。固体基盤としては通常
ガラス、石英、金属（金、白金）、プラスチック、シリ
コンウェハー等が用いられる。又、プロテインＡ単分子
膜を累積した累積膜は常法により得ることが出来、例え
ば水面上にプロテインＡの単分子膜を形成した後、水平
付着法あるいは垂直上下法により単分子膜を基盤上に移
し取るといった操作を繰り返すことにより累積膜を得る
ことが出来る。The antibody-immobilized membrane of the present invention can be obtained by applying the LB method by the following method, for example. That is, protein A is dissolved in a solvent, and this solution is dropped on the water surface in the LB device or is allowed to flow down to be developed. A protein A monolayer is formed at the gas-liquid interface. The film is compressed and held at a surface pressure lower than the pressure at which the film is broken and transferred onto a solid substrate. When the monomolecular film is accumulated, it is preferable to compress the surface pressure as high as possible. Here, the solvent is water or a buffer solution such as phosphoric acid, and the concentration of protein A is 0.05 to 1 g / l. The compression pressure on the membrane is usually 7 to 13 mN / m at which Protein A can maintain a single molecule state. As the solid substrate, glass, quartz, metal (gold, platinum), plastic, silicon wafer, etc. are usually used. In addition, a cumulative film obtained by accumulating protein A monolayers can be obtained by a conventional method. For example, after forming a protein A monolayer on the water surface, the monolayer is placed on the substrate by horizontal attachment method or vertical up / down method. A cumulative film can be obtained by repeating an operation such as transferring to.
【０００６】このようにして得られたプロテインＡ膜
を、抗体タンパク質溶液中に浸漬等の方法で接触させる
ことにより、プロテインＡ膜上に抗体タンパクを固定化
する。得られた抗体固定膜を例えば生理的リン酸緩衝液
で洗浄し、生化学的親和力以外で吸着している抗体を除
去する。これにより固体基盤上に固定化抗体超薄膜を得
ることが出来る。抗体タンパク質としては、哺乳動物の
免疫グロブリン（Ｉｇ）、特にＩｇＧのサブクラスであ
るＩｇＧ1，ＩｇＧ2、ＩｇＧ4等である。表１に固定化
抗体膜作成フローを示す。The protein A membrane thus obtained is brought into contact with an antibody protein solution by a method such as immersion to immobilize the antibody protein on the protein A membrane. The obtained antibody-immobilized membrane is washed with, for example, a physiological phosphate buffer to remove the adsorbed antibody other than the biochemical affinity. This makes it possible to obtain an immobilized antibody ultrathin film on a solid substrate. Antibody proteins include mammalian immunoglobulins (Ig), particularly IgG 1 , IgG 2 , IgG 4 which are subclasses of IgG. Table 1 shows a flow for preparing an immobilized antibody membrane.
【０００７】[0007]
【表１】 [Table 1]
【０００８】このように固定化された抗体固定膜は、プ
ロテインＡ・ＬＢ膜と抗体のＦｃ部位の結合により抗体
を基盤に固定しているため、抗体タンパクは変性を起こ
さず、抗原認識部位の活性を完全に保持したまま高い抗
体密度で基盤上に固定化されている。図１に本発明の抗
体固定膜の模式図を示す。抗体はプロテインＡ単分子膜
にＦｃ部位で結合し抗原認識部位であるＦａｂ部位が表
面に向いた状態で並んでいる。このことは抗原との反応
が速くしかも効率よく行う事が出来る。次にバイオセン
サーについて述べると、固定基盤上、例えば、石英基盤
上にプロテインＡ膜を積層し、抗体溶液中に浸漬し、該
抗体を固定化し、この抗体に対する抗原をあらかじめ蛍
光剤等で標識しておき、この標識抗原と測定対象物の抗
原とを競争反応させるか、または、標識抗原をあらかじ
めプロテインＡ上の固定化された抗体と反応させてお
き、測定対象物である抗原と交換反応をさせることによ
り、バイオセンサーを作製する。又、同様にして、本発
明の抗体固定膜を用いてバイオリアクター、バイオエレ
クトロニクスデバイス、免疫測定基盤を作成することが
できる。The thus-immobilized antibody-immobilized membrane immobilizes the antibody on the substrate by binding of the protein A / LB membrane and the Fc portion of the antibody, so that the antibody protein does not undergo denaturation and the antigen recognition site It is immobilized on a substrate with a high antibody density while completely retaining its activity. FIG. 1 shows a schematic diagram of the antibody-immobilized membrane of the present invention. The antibodies are bound to the protein A monolayer at the Fc site, and the Fab sites, which are the antigen recognition sites, are arranged in a state of facing the surface. This allows the reaction with the antigen to be rapid and efficient. Next, regarding the biosensor, a protein A film is laminated on a fixed substrate, for example, a quartz substrate, immersed in an antibody solution to immobilize the antibody, and the antigen for this antibody is labeled in advance with a fluorescent agent or the like. In advance, the labeled antigen and the antigen to be measured are allowed to undergo a competitive reaction, or the labeled antigen is reacted with the antibody immobilized on Protein A in advance, and the exchange reaction with the antigen to be measured is carried out. By doing so, a biosensor is produced. Similarly, a bioreactor, bioelectronic device, and immunoassay base can be prepared using the antibody-immobilized membrane of the present invention.
【０００９】[0009]
【作用】本発明の抗体固定膜は、基盤膜に対し生化学的
親和力で結合するため強固で高密度な抗体膜を得ること
が出来る。本発明の抗体固定膜では抗体のＦｃ部位とプ
ロテインＡ・ＬＢ膜とが結合するため、抗体を変えるこ
とにより種々の抗原に対する抗体膜を作ることが出来
る。そして、この抗体固定膜を用いてセンサーを作製し
た場合、抗体の抗原認識部位が外側を向いているため、
測定対象となる抗原と容易に反応することができ、した
がって得られるバイオセンサー、バイオリアクター、バ
イオエレクトロニクスデバイス、免疫測定基盤等は、優
れた作用効果を有する。The antibody-immobilizing membrane of the present invention binds to the base membrane with a biochemical affinity, so that a strong and high-density antibody membrane can be obtained. In the antibody-immobilized membrane of the present invention, since the Fc portion of the antibody binds to the protein A / LB membrane, it is possible to form antibody membranes against various antigens by changing the antibody. And when a sensor is produced using this antibody-immobilized membrane, the antigen recognition site of the antibody faces outward,
The biosensor, bioreactor, bioelectronic device, immunoassay platform, etc. that can easily react with the antigen to be measured have excellent effects.
【００１０】[0010]
実施例１．抗ヒト血清アルブミン抗体の固定化 プロテインＡ水溶液（０．５ｍｇ／ｍｌ）５０μｌをＬ
Ｂ膜製造装置の清浄な水面上にマイクロシリンジを用い
て展開させた。表面圧を１２ｍＮ／ｍに保ちプロテイン
Ａの単分子膜をステアリルトリクロルシランで疎水化処
理した無蛍光ガラス基盤上に２層積層した。この基盤を
抗ヒト血清アルブミン抗体の生理的リン酸緩衝溶液＊1
（１０ｍｇ／ｍｌ）中に１時間浸漬する。生理的リン酸
緩衝溶液で十分洗浄後、フルオレセインイソチオシアネ
ート標識したヒト血清アルブミン溶液１０~6〜１０~1ｍ
ｇ／ｍｌに１時間浸漬した。図２に示すように、ヒト血
清アルブミン濃度の増加と共に蛍光強度が増加した。こ
の結果は、抗ヒト血清アルブミン抗体は活性を十分保持
しプロテインＡ単分子膜上に固定化されていることを示
している。又、プロテインＡ単分子膜上に抗体タンパク
質が結合しているため固定化抗体膜を超薄膜の状態で作
製することが出来た。 *１：組成 ０．１５Ｍ ＮａＣｌ＋０．０１Ｍ リン酸
ナトリウム（ｐＨ７．０）。Example 1. Immobilization of anti-human serum albumin antibody 50 μl of protein A aqueous solution (0.5 mg / ml) was added to L
It was developed using a microsyringe on the clean water surface of the B film manufacturing apparatus. The surface pressure was kept at 12 mN / m, and two monolayers of protein A were laminated on a non-fluorescent glass substrate which was hydrophobized with stearyltrichlorosilane. Based on this basis, a physiological phosphate buffer solution of anti-human serum albumin antibody * 1
Immerse in (10 mg / ml) for 1 hour. After sufficiently washing with a physiological phosphate buffer solution, a human serum albumin solution labeled with fluorescein isothiocyanate 10 to 6 to 10 to 1 m
It was immersed in g / ml for 1 hour. As shown in FIG. 2, the fluorescence intensity increased with the increase of human serum albumin concentration. This result indicates that the anti-human serum albumin antibody retains the activity sufficiently and is immobilized on the protein A monolayer. Further, since the antibody protein is bound on the protein A monomolecular film, the immobilized antibody film could be produced in an ultrathin state. * 1: Composition: 0.15M NaCl + 0.01M sodium phosphate (pH 7.0).
【００１１】実施例２．ヒトＩｇＥセンサー プロテインＡ水溶液（０．５ｍｇ／ｍｌ）５０μｌをＬ
Ｂ膜製造装置の清浄な水面上にマイクロシリンジを用い
て展開させた。表面圧を１２ｍＮ／ｍに保ちプロテイン
Ａの単分子膜をステアリルトリクロルシランで疎水化処
理した無蛍光ガラス基盤上に２層積層した。この基盤を
抗ヒトＩｇＥ抗体の生理的リン酸緩衝溶液（５ｍｇ／ｍ
ｌ）中に１時間浸漬する。０．０５％Ｔｗｅｅｎ２０を
含む生理的リン酸緩衝溶液で十分洗浄後、フルオレセイ
ンイソチオシアネート標識したラットＩｇＥ溶液（０．
２ｍｇ／ｍｌ）と１０~6〜１０~3ｍｇ／ｍｌのヒトＩｇ
Ｅを含む人工血清（８％ウシ血清アルブミンを含む生理
的リン酸緩衝溶液）中に基盤を１時間浸漬した。図３に
示すようにＩｇＥ濃度が１０~3〜１０~6ｍｇ／ｍｌで蛍
光強度が直線的に変化し定量性のあることが判った。Embodiment 2. Human IgE sensor 50 μl of protein A aqueous solution (0.5 mg / ml) was added to L
It was developed using a microsyringe on the clean water surface of the B film manufacturing apparatus. The surface pressure was kept at 12 mN / m, and two monolayers of protein A were laminated on a non-fluorescent glass substrate which was hydrophobized with stearyltrichlorosilane. This substrate was used as a physiological phosphate buffer solution (5 mg / m 2) of anti-human IgE antibody.
Immerse in l) for 1 hour. After sufficient washing with a physiological phosphate buffer solution containing 0.05% Tween 20, the rat IgE solution labeled with fluorescein isothiocyanate (0.
2 mg / ml) and 10 ~ 6 ~10 ~ 3 mg / ml of human Ig
The substrate was immersed in artificial serum containing E (physiological phosphate buffer solution containing 8% bovine serum albumin) for 1 hour. As shown in FIG. 3, it was found that the fluorescence intensity changed linearly when the IgE concentration was 10 to 3 to 10 to 6 mg / ml, and that it was quantitative.
【００１２】[0012]
【発明の効果】プロテインＡ単分子膜上に抗体タンパク
質が結合しているため固定化抗体膜を超薄膜の状態で作
製することができ、得られた本発明の抗体固定膜は基盤
膜に対し生化学的親和力で結合しているため強固で高密
度のものとなる。又抗体を変えることにより種々の抗原
に対する抗体膜を容易に短時間に作ることができる。本
発明の抗体固定膜は抗体のＦｃ部位とプロテインＡ・Ｌ
Ｂ膜とが結合するため抗体の抗原認識部位が外側を向い
ており、そのため、この抗体固定膜を用いてセンサーを
作製した場合、測定対象となる抗原と容易に反応するこ
とが出来るという長所がある。従来、この膜のように抗
体の向きをコントロール（抗原認識部位を外側に向ける
事）することは出来なかった。又本抗体固定基盤を用い
たセンサーは従来の酵素反応等の測定のための時間を大
巾に短縮でき、操作性が極めて良好である。EFFECTS OF THE INVENTION Since the antibody protein is bound on the protein A monolayer, the immobilized antibody membrane can be prepared in an ultrathin state, and the obtained antibody-immobilized membrane of the present invention is used for the substrate membrane. Since they are bound by biochemical affinity, they are strong and dense. Also, by changing the antibody, it is possible to easily form an antibody film against various antigens in a short time. The antibody-immobilized membrane of the present invention comprises an antibody Fc portion and protein A / L.
Since the antigen recognition site of the antibody faces outward because it binds to the B membrane, the advantage of being able to easily react with the antigen to be measured when a sensor is prepared using this antibody-immobilized membrane is is there. Conventionally, it was not possible to control the direction of the antibody (directing the antigen recognition site to the outside) like this membrane. Further, the sensor using the antibody-immobilized substrate can greatly reduce the time required for the conventional measurement of enzyme reaction and the like, and has excellent operability.
【図１】本発明の抗体固定膜の模式図である。FIG. 1 is a schematic diagram of an antibody-immobilized membrane of the present invention.
【図２】本発明の抗体固定膜によるヒト血清アルブミン
の濃度と蛍光強度との関係を示すグラフである。FIG. 2 is a graph showing the relationship between the concentration of human serum albumin by the antibody-immobilized membrane of the present invention and the fluorescence intensity.
【図３】本発明の抗体固定膜を用いたヒトＩｇＥセンサ
ーのＩｇＥ濃度に対する蛍光強度変化を示すグラフであ
る。FIG. 3 is a graph showing changes in fluorescence intensity with respect to IgE concentration of a human IgE sensor using the antibody-immobilized membrane of the present invention.
Claims (4)
し、固体表面に写し取った後、プロテインＡの膜上に抗
体タンパク質を作用させることにより、抗体タンパクを
固定化する方法。1. A method of immobilizing an antibody protein by dropping an aqueous solution of protein A onto the surface of the water, transferring it onto a solid surface, and then allowing the antibody protein to act on the membrane of protein A.
サー、バイオリアクターもしくはバイオエレクトロニク
スデバイス。3. A biosensor, bioreactor or bioelectronic device using the antibody-immobilized membrane according to claim 2.
定法。4. An enzyme immunoassay method using the antibody-immobilized membrane according to claim 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4003257A JP2922040B2 (en) | 1992-01-10 | 1992-01-10 | Method for immobilizing antibody protein with protein A molecular membrane and antibody immobilized membrane |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4003257A JP2922040B2 (en) | 1992-01-10 | 1992-01-10 | Method for immobilizing antibody protein with protein A molecular membrane and antibody immobilized membrane |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH05273212A true JPH05273212A (en) | 1993-10-22 |
| JP2922040B2 JP2922040B2 (en) | 1999-07-19 |
Family
ID=11552420
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4003257A Expired - Fee Related JP2922040B2 (en) | 1992-01-10 | 1992-01-10 | Method for immobilizing antibody protein with protein A molecular membrane and antibody immobilized membrane |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2922040B2 (en) |
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