JPH047760B2 - - Google Patents
Info
- Publication number
- JPH047760B2 JPH047760B2 JP19722186A JP19722186A JPH047760B2 JP H047760 B2 JPH047760 B2 JP H047760B2 JP 19722186 A JP19722186 A JP 19722186A JP 19722186 A JP19722186 A JP 19722186A JP H047760 B2 JPH047760 B2 JP H047760B2
- Authority
- JP
- Japan
- Prior art keywords
- antibody protein
- antibody
- water
- monolayer
- aqueous phase
- 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.)
- Expired
Links
- 102000004169 proteins and genes Human genes 0.000 claims description 30
- 108090000623 proteins and genes Proteins 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 11
- 239000000758 substrate Substances 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 10
- 230000003100 immobilizing effect Effects 0.000 claims description 8
- 239000008346 aqueous phase Substances 0.000 claims description 6
- 239000002356 single layer Substances 0.000 claims description 6
- 150000002148 esters Chemical class 0.000 claims description 5
- 239000013554 lipid monolayer Substances 0.000 claims description 4
- 229910052751 metal Inorganic materials 0.000 claims description 4
- 239000002184 metal Substances 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 125000004432 carbon atom Chemical group C* 0.000 claims description 3
- 150000004668 long chain fatty acids Chemical class 0.000 claims description 3
- 230000005501 phase interface Effects 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 20
- 239000012528 membrane Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 235000021355 Stearic acid Nutrition 0.000 description 7
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 7
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 7
- 239000008117 stearic acid Substances 0.000 description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 3
- 229910001626 barium chloride Inorganic materials 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 230000007480 spreading Effects 0.000 description 3
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 2
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 235000021314 Palmitic acid Nutrition 0.000 description 2
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 2
- 229910052793 cadmium Inorganic materials 0.000 description 2
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- UKMSUNONTOPOIO-UHFFFAOYSA-N docosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCC(O)=O UKMSUNONTOPOIO-UHFFFAOYSA-N 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- VKOBVWXKNCXXDE-UHFFFAOYSA-N icosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCC(O)=O VKOBVWXKNCXXDE-UHFFFAOYSA-N 0.000 description 2
- 229940027941 immunoglobulin g Drugs 0.000 description 2
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 2
- 235000015497 potassium bicarbonate Nutrition 0.000 description 2
- 239000011736 potassium bicarbonate Substances 0.000 description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 2
- 229940086066 potassium hydrogencarbonate Drugs 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- TWJNQYPJQDRXPH-UHFFFAOYSA-N 2-cyanobenzohydrazide Chemical compound NNC(=O)C1=CC=CC=C1C#N TWJNQYPJQDRXPH-UHFFFAOYSA-N 0.000 description 1
- 235000021357 Behenic acid Nutrition 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 1
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 235000021360 Myristic acid Nutrition 0.000 description 1
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 description 1
- VCUFZILGIRCDQQ-KRWDZBQOSA-N N-[[(5S)-2-oxo-3-(2-oxo-3H-1,3-benzoxazol-6-yl)-1,3-oxazolidin-5-yl]methyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C1O[C@H](CN1C1=CC2=C(NC(O2)=O)C=C1)CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F VCUFZILGIRCDQQ-KRWDZBQOSA-N 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 150000001342 alkaline earth metals Chemical class 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229940116226 behenic acid Drugs 0.000 description 1
- 239000003012 bilayer membrane Substances 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229940015047 chorionic gonadotropin Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
【発明の詳細な説明】
＜産業上の利用分野＞
本発明は抗体タンパクを固定化する方法に関す
る。更に詳しくは、水溶性抗体タンパクを、その
抗原−抗体反応活性を完全に保持しながら固体基
板上に該タンパクを高密度に固定化する方法に関
する。DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for immobilizing antibody proteins. More specifically, the present invention relates to a method for immobilizing a water-soluble antibody protein at high density on a solid substrate while completely retaining its antigen-antibody reaction activity.
＜背景及び従来技術＞
抗体タンパクの抗原−抗体反応を利用した診断
技術に於て、抗体タンパクを固体基板上に固定化
する方法がこれまで数多く提案されてきた。これ
らの内、あるものは、抗体タンパクのアミノ基又
はカルボキシル基と反応又は吸着結合できる官能
基を有する固体表面に固定化するものであるが、
この場合、固定化のための反応条件の設定によつ
ては、抗体タンパクの変性や、非特異的反応によ
る活性部位の失活が起りやすいという問題点があ
つた。また、親水性ゲルの中に抗体タンパクを抱
き込ませて、固体基板上に固定化する方法も可能
性のある方法であるが、この場合、抗原タンパク
がゲル中の抗体タンパクに接近でき難くなり、抗
原−抗体反応を利用した診断材料としての感度低
下を招き易い。こうした方法に対して近年、水面
上に脂質膜を展開し、これに水中から水溶性酵素
（例、カタラーゼ，フエリチン，ヘミグロビン，
グルコースオキシダーゼ，etc.）や抗体タンパク
［免疫グロブリンＧ（IgG），IgE，etc］を吸着固
定化する試みが報告されている［バイオキミカ・
エト・バイオフイジカ・アクタ（Biochem.
Biophys.Acta.）225巻，382（1971）やジヤーナ
ル・オブ・セルラー・バイオケミストリー（J.
Cell.Biochem.）29，239（1985）等参照］。<Background and Prior Art> In diagnostic techniques that utilize antigen-antibody reactions of antibody proteins, many methods have been proposed to date for immobilizing antibody proteins on solid substrates. Among these, some are immobilized on a solid surface that has a functional group that can react or adsorb and bond with the amino group or carboxyl group of the antibody protein.
In this case, depending on the setting of the reaction conditions for immobilization, there was a problem that denaturation of the antibody protein and deactivation of the active site due to non-specific reactions were likely to occur. Another possibility is to envelop the antibody protein in a hydrophilic gel and immobilize it on a solid substrate, but in this case, it becomes difficult for the antigen protein to access the antibody protein in the gel. , which tends to result in a decrease in sensitivity as a diagnostic material that utilizes antigen-antibody reactions. In response to these methods, in recent years a lipid membrane has been developed on the water surface, and water-soluble enzymes (e.g., catalase, ferritin, hemiglobin, etc.) are extracted from the water.
Attempts to adsorb and immobilize glucose oxidase, etc.) and antibody proteins [immunoglobulin G (IgG), IgE, etc.] have been reported [Biochimica
Biochem.
Biophys.Acta.) vol. 225, 382 (1971) and Journal of Cellular Biochemistry (J.
Cell.Biochem.) 29 , 239 (1985), etc.].
これらは確かにタンパクを、その活性を保持し
たまま固定化する優れた方法であるが、この方法
に於て従来用いられてきた脂質膜は、ステアリン
酸、アラキン酸及びジパルミトイルホスフアチジ
ルコリン（DPPC）等の如く、水に対して可溶性
（少くとも、1μｇ／１水の溶解度）のもの、又
は水中で二分子膜ベシクルを形成しやすいもの
（DPPC等）であつた。従つて、水面上で該膜に
吸着された抗体タンパクは、時間が経つにつれ、
脂質膜と共に再び水中に再溶解したり、一旦固体
基板上に累積した後でも抗原水溶液に浸漬した
際、脱離しやすいという問題点を有していた。 These are certainly excellent methods for immobilizing proteins while retaining their activity, but the lipid membranes conventionally used in this method are stearic acid, arachidic acid, dipalmitoylphosphatidylcholine ( They were those that were soluble in water (at least 1 μg/water solubility), such as DPPC), or those that easily formed bilayer membrane vesicles in water (DPPC, etc.). Therefore, over time, the antibody protein adsorbed to the membrane on the water surface will
It has a problem that it is easily detached when it is redissolved in water together with the lipid membrane or when it is immersed in an aqueous antigen solution even after it has been accumulated on a solid substrate.
＜発明の目的＞
本発明者らは、かかる従来技術の欠点を克服
し、高い抗原−抗体反応活性の保持率と、高密度
な抗体タンパクの固定化を可能にすべく鋭意検討
の結果、本発明に到達したものである。<Purpose of the Invention> As a result of intensive studies, the present inventors have developed the present invention in order to overcome the drawbacks of such conventional techniques and to enable high retention of antigen-antibody reaction activity and high-density immobilization of antibody proteins. This invention has been achieved.
＜発明の開示＞
すなわち本発明は、水相面上に展開された炭素
原子数14〜23の長鎖脂肪酸の多価金属塩及び／又
はエステルの単分子膜に当該水相中に溶解した水
溶性抗体タンパク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ことを特徴とす
る脂質単分子膜により抗体タンパクを固定化する
方法である。<Disclosure of the Invention> That is, the present invention provides a monomolecular film of a polyvalent metal salt and/or ester of a long-chain fatty acid having 14 to 23 carbon atoms spread on the surface of an aqueous phase. A method for immobilizing an antibody protein with a lipid monolayer, characterized by forming an antibody protein-monolayer complex at the aqueous phase interface by contacting the antibody protein with a lipid monolayer, and layering the complex on a solid substrate. It is.
本発明でいう抗体タンパクとは、抗原−抗体反
応を起しうる水溶性タンパクの総称であり、その
分子中に抗原認識部位（Fabと略）と疎水性末端
部位（Fcと略）を有している。 The antibody protein used in the present invention is a general term for water-soluble proteins that can cause antigen-antibody reactions, and has an antigen recognition site (abbreviated as Fab) and a hydrophobic terminal site (abbreviated as Fc) in its molecule. ing.
かかる抗体タンパクの具体例としては、免疫グ
ロブリンＧ（IgGと略称），IgE，IgM及びこれら
の抗体，絨毛性性腺刺激ホルモン（HCG）抗体、
ガン胎児性抗原（CEA）抗体等があげられる。 Specific examples of such antibody proteins include immunoglobulin G (abbreviated as IgG), IgE, IgM and their antibodies, chorionic gonadotropin (HCG) antibodies,
Examples include carcinoembryonic antigen (CEA) antibodies.
これらの抗体タンパクの固定化にあたつては、
Fab部分を変性しないようにすることが肝要であ
るが、前述の如く従来の化学反応による固定化の
場合Fab部分も反応に関与して抗体タンパクの活
性低下を招いていた。本発明においては抗体タン
パクは、後述の脂質単分子膜中にFc部位で疎水
的に相互作用しながら高密度にくみ込まれるか、
イオン的相互作用により免疫活性を高く保持した
まま単分子膜に吸着固定される。 When immobilizing these antibody proteins,
It is important to prevent the Fab portion from being denatured, but as mentioned above, in the case of immobilization by conventional chemical reactions, the Fab portion also participates in the reaction, resulting in a decrease in the activity of the antibody protein. In the present invention, the antibody protein is packed into a lipid monolayer (described later) at high density while interacting hydrophobically at the Fc site, or
Due to ionic interaction, it is adsorbed and immobilized on the monolayer while maintaining high immune activity.
上記の抗体タンパクを固定化するための脂質単
分子膜としては、水面上で固体上の凝縮単分子膜
を形成し、水に実質的に溶解しないものが好まし
い。そのようなものとして、炭素原子数14〜22の
長鎖脂肪酸の多価金属塩及び／又はエステルが用
いられる。このような脂肪酸としてはミリスチン
酸（C13H27COOH），パルミチン酸
（C15H31COOH），ステアリン酸
（C17H35COOH），アラギン酸（C19H39COOH），
ベヘン酸（C21H43COOH）が代表的なものとし
て挙げられる。従つてCnH2n＋1COM（ｎ＝14〜
22，Ｍ＝アルカリ土類金属、カドミウム、アルミ
ニウム等の多価金属イオン）で表わされる長鎖脂
肪酸の多価金属塩及び前記脂肪酸のメタノール又
はエタノールとのエステルである。 The lipid monomolecular film for immobilizing the antibody protein described above is preferably one that forms a solid condensed monomolecular film on the water surface and does not substantially dissolve in water. As such, polyvalent metal salts and/or esters of long-chain fatty acids having 14 to 22 carbon atoms are used. Such fatty acids include myristic acid (C 13 H 27 COOH), palmitic acid (C 15 H 31 COOH), stearic acid (C 17 H 35 COOH), aragic acid (C 19 H 39 COOH),
Behenic acid (C 21 H 43 COOH) is a typical example. Therefore, CnH2n+1COM (n=14~
22, M=polyvalent metal ions such as alkaline earth metals, cadmium, aluminum, etc.) and esters of the fatty acids with methanol or ethanol.
これらは例えばカルボン酸又はそのエステルの
状態でベンゼンやクロロホルム等の有機溶媒に溶
解させ、0.5〜1.5ミリモル／の溶液となしたの
ち、これを蒸溜水又は多価金属塩（例えば、塩化
バリウム、塩化カドミウム、塩化アルミニウム）
を含む、PH6.5〜7.5の水溶液表面上に展開するこ
とにより、本発明に用いる単分子膜となしうる。
次いで、該単分子膜を、その表面圧力が１〜
30mN（ミリＮ）／ｍになるように圧縮したのち、
そのままの圧縮条件で膜面下の水相に前記の抗体
タンパクを注入する。所定時間（通常、30分〜１
時間）、該抗体タンパクと水面上の単分子膜とを
接触させておくことにより、タンパクと該単分子
膜との複合化が完了する。この時点で抗体タンパ
クと単分子膜との複合体膜を、表面圧力30〜50ミ
リＮ／ｍにて再び圧縮したのち、固体基板上にラ
ングミユア・ブロジエツト法又は水平付着法（詳
細は新実験化学講座第18巻第439頁参照）により
一層又は複数層積層することができる。 These are dissolved in an organic solvent such as benzene or chloroform in the form of a carboxylic acid or its ester to form a solution of 0.5 to 1.5 mmol/ml, and then dissolved in distilled water or a polyvalent metal salt (e.g., barium chloride, chloride, etc.). cadmium, aluminum chloride)
A monomolecular film used in the present invention can be obtained by spreading it on the surface of an aqueous solution containing PH6.5 to 7.5.
Next, the monomolecular film is coated with a surface pressure of 1 to
After compressing to 30mN (milliN)/m,
The above antibody protein is injected into the aqueous phase below the membrane surface under the same compression conditions. Specified time (usually 30 minutes to 1
time), by keeping the antibody protein in contact with the monolayer on the water surface, the complexation of the protein and the monolayer is completed. At this point, the composite film of the antibody protein and the monolayer is compressed again at a surface pressure of 30 to 50 mmN/m, and then deposited on a solid substrate using the Langmiur-Blodget method or the horizontal adhesion method (see New Experimental Chemistry for details). (Refer to Volume 18, Page 439 of Lectures), it is possible to laminate one or more layers.
その際の抗体タンパクの固体基板上への固定化
量は、積層時の水面展開膜の基板への転移比及び
UV−吸収スペクトル強度より算出される。 At this time, the amount of antibody protein immobilized on the solid substrate is determined by the transfer ratio of the water surface-deployed membrane to the substrate during stacking and
Calculated from UV-absorption spectrum intensity.
かくして得られた基板上の複合体は、抗体タン
パクの抗原−抗体反応活性を酵素免疫診断法
（EIA）により求めた結果、すぐれた活性を示す
ものであつた。 The thus obtained complex on the substrate showed excellent activity when the antigen-antibody reaction activity of the antibody protein was determined by enzyme immunodiagnosis (EIA).
以下、実施例により本発明を更に詳しく説明す
る。 Hereinafter, the present invention will be explained in more detail with reference to Examples.
実施例 １
ステアリン酸（C17H35CO2H）10mgを25mlの蒸
留クロロホルムに溶解し、562cm2の水槽表面積を
有する表面圧−面積曲線（以下、π−Ａ曲線と略
す）測定用水槽に張つた塩化バリウム３×10-5M
炭酸水素カリウム４×10-4M混合水溶液上にウル
トラマイクロピペツトを用いて、上記溶液150μ
を徐々に滴下した。滴下終了後水面展開膜を５
分間静置してからヒトIgG水溶液を濃度0.03mg／
mlになるように水槽中へ注入した。１時間静置後
仕切板の移動を開始しπ−Ａ曲線を測定した。そ
の結果、π−Ａ曲線はステアリン酸を単独展開し
た場合に比して膨脹したものとなつた。Example 1 10 mg of stearic acid (C 17 H 35 CO 2 H) was dissolved in 25 ml of distilled chloroform and placed in a water tank for measuring a surface pressure-area curve (hereinafter abbreviated as π-A curve) having a tank surface area of 562 cm 2 . Barium chloride 3×10 -5 M
Using an ultramicropipette, pipet 150μ of the above solution onto a 4×10 -4 M mixed aqueous solution of potassium hydrogen carbonate.
was gradually added dropwise. After dropping, spread the membrane on the water surface for 5 minutes.
After standing for a minute, add a human IgG aqueous solution to a concentration of 0.03 mg/
ml was injected into the aquarium. After standing still for 1 hour, the partition plate was moved and the π-A curve was measured. As a result, the π-A curve was expanded compared to when stearic acid was developed alone.
この水面展開膜を30ミリＮ／ｍの表面圧力下で
圧縮しながら、疎水化処理（シランカツプリング
処理）を施した石英板上に垂直浸漬引き上げ法
（以降LB法と略）によつて２層累積した。 While compressing this water surface spread membrane under a surface pressure of 30 mmN/m, it was placed on a quartz plate that had been subjected to hydrophobization treatment (silane coupling treatment) by vertical dipping and pulling up (hereinafter abbreviated as LB method). The layers were accumulated.
平均累積比は0.74であつた。紫外吸収スペクト
ル測定によると、石英板上のヒトIgGの被覆率は
3.0×10-8mol／m2であつた。 The average cumulative ratio was 0.74. According to ultraviolet absorption spectrometry, the coverage of human IgG on the quartz plate is
It was 3.0×10 -8 mol/m 2 .
次いでこの累積膜にペルオキシターゼ標識抗ヒ
トIgG抗体（20μ／ml）液を作用させた後、オ
ルトフエニレンジアミン、過酸化水素混合溶液中
に浸漬した処、呈色（褐色）が認められ、累積膜
中のヒトIgGの抗原活性が保持されていることが
わかつた。 Next, a peroxidase-labeled anti-human IgG antibody (20μ/ml) solution was applied to this accumulated film, and when it was immersed in a mixed solution of orthophenylenediamine and hydrogen peroxide, coloration (brown) was observed, and the accumulated film was It was found that the antigenic activity of human IgG in the protein was retained.
比較例
実施例１と同様に調製したステアリン酸溶液
150μをπ−Ａ測定用水槽に張つた蒸溜水上に
徐々に滴下した。滴下終了後水面展開膜を５分間
静置してから表面圧30ミリＮ／ｍに保つよう仕切
板を移動させた処、水面展開膜の面積は減少しつ
づけ、ステアリン酸の蒸溜水中への溶解が見られ
た。Comparative Example Stearic acid solution prepared in the same manner as Example 1
150μ was gradually dropped onto distilled water placed in a water tank for π-A measurement. When the water surface spreading membrane was allowed to stand still for 5 minutes after the dropping was completed, and the partition plate was moved to maintain the surface pressure at 30 mmN/m, the area of the water surface spreading membrane continued to decrease, and the dissolution of stearic acid in distilled water increased. It was observed.
実施例 ２
実施例１に於て、ステアリン酸の代りに、パル
ミチン酸を用いて同様に塩化バリウムと炭酸水素
カリウムの稀薄水溶液表面上に展開した後、30分
静置後、30ミリＮ／ｍの表面圧下で圧縮しなが
ら、石英ガラス基板に２層累積した。その際の累
積比は0.72であつた。このものは、実施例１と同
様にして抗原活性を評価した処、水溶液状態での
抗原活性と同等の活性を有していた。Example 2 In Example 1, palmitic acid was used instead of stearic acid and spread on the surface of a dilute aqueous solution of barium chloride and potassium hydrogen carbonate. The two layers were deposited on a quartz glass substrate while being compressed under a surface pressure of . The cumulative ratio at that time was 0.72. The antigen activity of this product was evaluated in the same manner as in Example 1, and it was found to have an antigen activity equivalent to that in the aqueous solution state.
Claims (1)
鎖脂肪酸の多価金属塩及び／又はエステルの単分
子膜に当該水相中に溶解した水溶性抗体タンパク
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
板上に積層することを特徴とする脂質単分子膜に
より抗体タンパクを固定化する方法。1. By bringing a water-soluble antibody protein dissolved in the aqueous phase into contact with a monomolecular film of a polyvalent metal salt and/or ester of a long-chain fatty acid with 14 to 22 carbon atoms spread on the surface of the aqueous phase. 1. A method for immobilizing an antibody protein using a lipid monolayer, which comprises forming an antibody protein-monolayer complex at an aqueous phase interface and layering the complex on a solid substrate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19722186A JPS6354400A (en) | 1986-08-25 | 1986-08-25 | Immobilization of antibody protein with lipid monomolecular film |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19722186A JPS6354400A (en) | 1986-08-25 | 1986-08-25 | Immobilization of antibody protein with lipid monomolecular film |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6354400A JPS6354400A (en) | 1988-03-08 |
| JPH047760B2 true JPH047760B2 (en) | 1992-02-12 |
Family
ID=16370848
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP19722186A Granted JPS6354400A (en) | 1986-08-25 | 1986-08-25 | Immobilization of antibody protein with lipid monomolecular film |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6354400A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU638278B2 (en) * | 1989-09-07 | 1993-06-24 | Matsushita Electric Industrial Co., Ltd. | Washing machine |
| JP5891951B2 (en) * | 2012-05-28 | 2016-03-23 | トヨタ紡織株式会社 | Vehicle seat |
-
1986
- 1986-08-25 JP JP19722186A patent/JPS6354400A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6354400A (en) | 1988-03-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3713516B2 (en) | Sensors based on polymer transformations | |
| US4987032A (en) | Functional organic thin film and method of manufacture thereof | |
| US6291155B1 (en) | Self assembly of sensor membranes | |
| US6241863B1 (en) | Amperometric biosensors based on redox enzymes | |
| JPH08505123A (en) | Improved sensor membrane | |
| JPH04501605A (en) | Detection surfaces capable of selective biomolecular interactions for use in biosensor systems | |
| JPH03505222A (en) | Method for immobilizing proteins, peptides, coenzymes, etc. on carriers | |
| WO1986004095A1 (en) | Preparation of reagents | |
| FR2534486A1 (en) | SURFACE GRAFTED PARTICLE SUPPORT, PROCESS FOR PREPARING THE SAME AND ADSORBENTS FOR AFFINITY CHROMATOGRAPHY INCORPORATING THE SAME, AND USE THEREOF, IN PARTICULAR IN BIOLOGY | |
| Yang et al. | Biomembrane mimetic surfaces by phospholipid self-assembled monolayers on silica substrates | |
| JPH0561279B2 (en) | ||
| JPH047760B2 (en) | ||
| Rodrigues et al. | Cellulase and alcohol dehydrogenase immobilized in Langmuir and Langmuir–Blodgett films and their molecular-level effects upon contact with cellulose and ethanol | |
| EP0451763B1 (en) | Peptide derivatives with amphiphatic properties, intermediates thereof and liposomes and films comprising said derivatives | |
| US6316273B1 (en) | Biosensor for detection of small molecule analytes | |
| US5879878A (en) | Method of producing a first layer electrode membrane for a biosensor | |
| JPH047759B2 (en) | ||
| FR2550799A1 (en) | ENZYME-MARKED COMPOUND, PROCESS FOR PREPARING THE SAME AND USE THEREOF IN ENZYMOIMMUNOLOGY | |
| JPS60155129A (en) | Preparation of immobilized antigen or antibody | |
| JP2614905B2 (en) | Immunosensor | |
| JPH0473749B2 (en) | ||
| Damrongchai et al. | Calcium responsive two-dimensional molecular assembling of lipid-conjugated calmodulin | |
| JPH02272081A (en) | Functional organic thin film | |
| US4950405A (en) | Functional thin organic membrane | |
| JP3509142B2 (en) | Reactive liposomes and forming agents |