JPH02207798A - Monoclonal antibody to ganglioside and hybridoma capable of producing antibody thereof - Google Patents
Monoclonal antibody to ganglioside and hybridoma capable of producing antibody thereofInfo
- Publication number
- JPH02207798A JPH02207798A JP1027507A JP2750789A JPH02207798A JP H02207798 A JPH02207798 A JP H02207798A JP 1027507 A JP1027507 A JP 1027507A JP 2750789 A JP2750789 A JP 2750789A JP H02207798 A JPH02207798 A JP H02207798A
- Authority
- JP
- Japan
- Prior art keywords
- monoclonal antibody
- ganglioside
- neuac
- antibody
- antigen
- 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.)
- Pending
Links
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 9
- 150000002270 gangliosides Chemical class 0.000 title abstract description 22
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- SQVRNKJHWKZAKO-LUWBGTNYSA-N N-acetylneuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-LUWBGTNYSA-N 0.000 abstract description 24
- 229940060155 neuac Drugs 0.000 abstract description 24
- CERZMXAJYMMUDR-UHFFFAOYSA-N neuraminic acid Natural products NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO CERZMXAJYMMUDR-UHFFFAOYSA-N 0.000 abstract description 24
- 239000000427 antigen Substances 0.000 abstract description 19
- 102000036639 antigens Human genes 0.000 abstract description 19
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- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 description 1
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- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
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- WPIHMWBQRSAMDE-YCZTVTEBSA-N beta-D-galactosyl-(1->4)-beta-D-galactosyl-N-(pentacosanoyl)sphingosine Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCC(=O)N[C@@H](CO[C@@H]1O[C@H](CO)[C@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O)[C@H](O)\C=C\CCCCCCCCCCCCC WPIHMWBQRSAMDE-YCZTVTEBSA-N 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、ガングリオシドに対するモノクローナル抗体
及びその抗体を産生ずるハイブリドーマに関する。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to monoclonal antibodies against gangliosides and hybridomas producing the antibodies.
（従来の技術および発明が解決しようとする課題）ガン
グリオシド（シアル酸含有スフィンゴ糖脂質）は微量で
はあるが、すべての高等動物細胞に存在する。現在まで
に構造解析された分子種は、５０種を超え、その分布は
、機能細胞の種類、動物種により特徴あるパターンを示
す。よって、その存在意義、生物学的役割の解明は現在
量も期待される課題の一つである。■型糖鎖をもつα２
−３シアリルバラグロボシド（ＩＶ’（ＮｅｕＡｃ）ｎ
Ｌｃ４Ｃｅｒ或はＮ−アセチルノイラミニルネオラクト
テトラオシルセラミド）は、ヒト赤血球、脳、末梢神経
等に存在するが、正常ヒト、組織中における存在は明か
でない。このガングリオシドは、本発明者により、ヒト
及びトリのインフルエンザウィルスへマグルチニン（Ｈ
ｌ、Ｈ２，Ｈ３亜型）に対する標的細胞膜上の共通の受
容体として機能していることが明らかにされた（Ｊ、旧
ｏ１． Ｃｈｅｍ、。(Prior Art and Problems to be Solved by the Invention) Gangliosides (sialic acid-containing glycosphingolipids) are present in all higher animal cells, albeit in trace amounts. More than 50 molecular species have been structurally analyzed to date, and their distribution shows distinctive patterns depending on the type of functional cell and animal species. Therefore, elucidating the significance of its existence and its biological role is one of the challenges that is expected to be solved even in terms of the current amount. α2 with type sugar chain
-3 Sialylvala globoside (IV'(NeuAc)n
Lc4Cer (or N-acetylneuraminyl neolactotetraosylceramide) exists in human red blood cells, brain, peripheral nerves, etc., but its presence in normal human tissues is unclear. This ganglioside has been developed by the present inventors into human and avian influenza virus hemagglutinin (H
It was revealed that it functions as a common receptor on the target cell membrane for 1, H2, and H3 subtypes (J, former o1. Chem.
２６１、１７０５７−１７０６１（１９８６）、 Ｂｉ
ｏｃｈｉｍ、 Ｂｉｏｐｈｙｓ、 Ａｃｔａ、 ９０３
．４１７−４２４（１９８７））。インフルエンザウィ
ルス膜にあるヘマグルチニン糖タンパク質は、ウィルス
の吸着や、膜融合を介する細胞内侵入にかかわる受容体
を認識する分子として機能するが、ウィルス特異抗厘と
して、感染防御上重要である。261, 17057-17061 (1986), Bi
ochim, Biophys, Acta, 903
．． 417-424 (1987)). The hemagglutinin glycoprotein in the influenza virus membrane functions as a molecule that recognizes receptors involved in virus adsorption and intracellular entry through membrane fusion, and is important for infection defense as a virus-specific antibody.
これをコードする遺伝子は非常に変異しやすく、それに
伴う抗原性の変異は、従来のワクチン効果を極めて困難
なものとしている。β２−３シアリルパラグロボシド（
ＩＶ”（ＮｅｕＡｃ）ｎＬｃ４Ｃｅｒ）は、多くのヒト
インフルエンザウィルスｃＡ型（Ｈｌ、Ｈ２゜Ｈ３亜型
）、Ｂ型］ヘマグルチニンが共通して認識する受容体で
あり、これにより、ウィルスの赤血球凝集、溶血が阻止
される。従ってこのガングリオシドは、ヒトインフルエ
ンザウィルスに対する広域性の高い抗ウィルス薬となり
うる。さらに、その抗イデイオタイプ（σ２−３シアリ
ルパラグロボシドに対する抗体の超可置部を認識した抗
体）は、β２−３シアリルパラグロボシドと同じ三次元
イメージを持つため、広域性の高い抗ウイルスタンパク
質として機能する。従って、β２−３シアリルパラグロ
ボシドに対する抗体は、従来のインフルエンザワクチン
とは異なる全く新しい広域インフルエンザワクチンとし
て使用できうる。The gene encoding this is highly mutable, and the accompanying antigenic variation makes it extremely difficult for conventional vaccines to be effective. β2-3 sialylparagloboside (
IV'' (NeuAc)nLc4Cer) is a receptor that is commonly recognized by many human influenza viruses type cA (Hl, H2゜H3 subtypes), type B] hemagglutinin, and thereby inhibits hemagglutination and hemolysis of the virus. Therefore, this ganglioside can be a broad-spectrum antiviral drug against human influenza virus.Furthermore, its anti-idiotype (an antibody that recognizes the superpositionable region of the antibody to σ2-3 sialylparagloboside) , has the same three-dimensional image as β2-3 sialylparagloboside, so it functions as a highly broad-spectrum antiviral protein. Therefore, antibodies against β2-3 sialylparagloboside are completely new and different from conventional influenza vaccines. It can be used as a broad-spectrum influenza vaccine.
一方、Ｉ型糖鎖を持つＩＶ’（ＮｅｕＡｃ）Ｌｃ４Ｃｅ
ｒ （Ｎ −アセチルノイラミニルラクトテトラオシル
セラミド）は、最近ヒト胎便中に存在することが報告さ
れ（ＦＥＢＳ Ｌａｔｔｅｒ、 １９８．６６−７０（
１９８６））、癌胎児性抗原と考えられている。又ヒト
大腸癌に対して作られたモノクローナル抗体（Ｃ−５０
）は、ｒｌ（ＮｅｕＡｃ）Ｌｃ４Ｃｅｒを認識している
（ＦＥＢＳ Ｌｅｔｔｅｒ、 １８２．３９８−４０２
（１９８５）、 Ｂｉｏｃｈｉｍ、 Ｂｉｏｐｈｙｓ、
Ａｃｔａ、 ８３４．１１０−１１７（１９８５））
。On the other hand, IV'(NeuAc)Lc4Ce with type I sugar chain
r (N-acetylneuraminyl lactotetraosylceramide) was recently reported to exist in human meconium (FEBS Latter, 198.66-70).
(1986)) and is considered a carcinoembryonic antigen. In addition, a monoclonal antibody (C-50) made against human colon cancer
) recognizes rl(NeuAc)Lc4Cer (FEBS Letter, 182.398-402
(1985), Biochim, Biophys,
Acta, 834.110-117 (1985))
.
このような経過を見るとＩＶ”（ＮａｕＡｃ）ｎＬｃ４
Ｃｅｒ。Looking at this progress, IV”(NauAc)nLc4
Cer.
及びＶ’ （ＮｅｕＡｃ）Ｌｃ４Ｃｅｒを特異的に認識
するモノクローナル抗体は、これらの抗原の生体におけ
る超微量解析、ヒト癌の診断、インフルエンザ広域ワク
チンの開発等にとって重要であるが、これまでガングリ
オシドの抗原性が弱く、得られていなかった。そのため
、このような特異的な抗体を産生ずるハイプリドーマの
樹立および樹立法が待ち望まれていた。 現在までに、
ＩＶ’（ＮｅｕＡｃ）ｎＬ４Ｃｅｒのみ確認するモノク
ロール抗体は得られていない。又ＩＶ”（ＮｅｕＡｃ）
ｎＬｃ４ｃｅｒ及びＩＶ’（ＮｅｕＡｃ）Ｌｃ４Ｃｅｒ
の両者を特異的に認識するモノクロナール抗体も得られ
ていない。Monoclonal antibodies that specifically recognize V' (NeuAc)Lc4Cer and V' (NeuAc)Lc4Cer are important for ultra-trace analysis of these antigens in living organisms, diagnosis of human cancer, and development of broad-spectrum influenza vaccines. was weak and not obtained. Therefore, the establishment and method for establishing hybridomas that produce such specific antibodies have been awaited. from now on,
A monoclonal antibody that only confirms IV'(NeuAc)nL4Cer has not been obtained. Mata IV” (NeuAc)
nLc4cer and IV'(NeuAc)Lc4Cer
A monoclonal antibody that specifically recognizes both has not yet been obtained.
（課題を解決するための手段）
ガングリオシドは、その抗原性が弱いことが知られてい
る。本発明者は、ガングリオシド抗原のセラミド部分を
リポソーム膜に埋め込むことにより抗原性を上げること
に成功し、さらに、これを自然抗原により感作されてい
る可能性の高い２２週令マウスに免疫することにより、
高い力価のモノクローナル抗体を得ることができた。本
発明にかかわるモノクローナル抗体（ＳＫ−８）は次の
ような性質を有する。(Means for solving the problem) Gangliosides are known to have weak antigenicity. The present inventor succeeded in increasing antigenicity by embedding the ceramide part of a ganglioside antigen into a liposome membrane, and further succeeded in immunizing 22-week-old mice, which are likely to have been sensitized by natural antigens, with this. According to
We were able to obtain high titer monoclonal antibodies. The monoclonal antibody (SK-8) according to the present invention has the following properties.
（１） Ｉ Ｖ ”（ＮｅｕＡｃ）ｎＬｃ４ｃｓｒ、及
びＩＩ（ＮｅｕＡｃ）Ｌｃ４Ｃｅｒのいずれをも認識す
る。即ち、ＮａｕＡｃａ２−３Ｇａｌβ１−４（３）Ｇ
１ｃＮＡｃβｌ−３Ｇａｌβ１−４Ｇ１ｃβ１−Ｃｅｒ
ａｍｉｄｅで示される構造を認識する。(1) Recognizes both I V ”(NeuAc)nLc4csr and II(NeuAc)Lc4Cer. That is, NauAca2-3Galβ1-4(3)G
1cNAcβl-3Galβ1-4G1cβ1-Cer
Recognize the structure indicated by amide.
（２）Ｎ−アセチルノイラミン酸を含む次のガングリオ
シド、ＩＶ’（ＮｅｕＡｃ）ｎＬｃ４Ｃｅｒ（ａ ２−
６シアリルパラグロボシド又はＮｅｕＡｃ ａ ２−６
Ｇａｌβｌ−４ＧｌｃＮＡｃβ１−３Ｇａｌβ１−Ｃｅ
ｒ）、Ｖｌ３（ＮｅｕＡｃ）ｎＬｃ６Ｃｅｒ（ＮｅｕＡ
ｃ ａ ２−３Ｇａｌβ１−４ＧｌｃＮＡｃβｌ−３Ｇ
ａｌβｌ−４Ｇ１ｃβ１−Ｃａｒ）、■−活性ガングリ
オシド（Ｇａｌ ａ ｌ−３Ｇａｌβｌ−４ＧｌｃＮＡ
ｃβ１−６（ＮｅｕＡｃ ａ ２−３Ｇａｌβｌ−４Ｇ
ｌｃＮＡｃβｌ−３）Ｇａｌβ１−４０１ｃＮＡｃβｌ
−３Ｇａｌβｌ−４Ｇｌｃβ１−Ｃａｒ）、ＧＭｌｂ（
ＮｅｕＡｃａ ２−３Ｇａｌβｌ−３ＧａｌＮＡｃβｌ
−４Ｇａｌβｌ−４Ｇ１ｃβ１−Ｃｅｒ）、Ｃ，Ｍｌａ
（Ｇａｌβ１−３ＧａｌＮＡｃβ１−４（ＮｅｕＡｃａ
２−３）Ｇａｌβｌ−４０１ｃβ１−Ｃｅｒ）、０Ｍ
２（ＧａｌＮＡｃβｌ−４（ＮｅｕＡｃａ ２−３）Ｃ
ａｌβ１４Ｇ１ｃβ１−Ｃａｒ）、ＧＤ２（ＧａｌＮＡ
ｃβｌ−４（ＮｅｕＡｃａ ２−８ＮｅｕＡｃａ ２−
３）Ｇａ１７？ｌ−４Ｇｔｃβ１−Ｃｅｒ）、ＧＤｌａ
（ＮｅｕＡｃａ ２−３）Ｇａｌβｌ−３ＧａｌＮＡｃ
７？ｌ−４（ＮｅｕＡｃａ ２−３）Ｇａｌβｌ−４Ｇ
ｌｃβ１−Ｃｅｒ）、ＧＤｌｂ（Ｇａｌβ１−３Ｇａ
ｌＮＡｃβ１−４（ＮａｕＡｃ２−８ＮｅｕＡｃａ ２
−３）Ｇａｌβｌ−４Ｇｌｃβ１−Ｃａｒ）、ＧＴｌｂ
（ＮｅｕＡｃａ ２−３Ｇａｌβｌ−３ＧａｌＮＡｃβ
ｌ−４（ＮａｕＡｃａ ２−８ＮｅｕＡｃａ ２−３）
Ｇａｌβｌ−４ＧＩｃβ１−Ｃｅｒ）、０Ｍ３（Ｎｅｕ
Ａｃａ ２−３Ｇａｌβｌ−４Ｇｌｃβ１−Ｃａｒ）、
ＧＤ３（ＮｅｕＡｃａ ２−８ＮｅｕＡｃａ ２−３Ｇ
ａｌβｌ−４０ＩＣβ１−Ｃｅｒ）、ＧＴ３（ＮｅｕＡ
ｃａ ２−８ＮｅｕＡｃａ ２−８ＮｅｕＡｃａ ２３
Ｇａ ｌβｌ−４ＧＩｃβ１−Ｃｅｒ）とは反応しない
。(2) The next ganglioside containing N-acetylneuraminic acid, IV'(NeuAc)nLc4Cer(a2-
6 Sialylparagloboside or NeuAc a 2-6
Galβl-4GlcNAcβ1-3Galβ1-Ce
r), Vl3(NeuAc)nLc6Cer(NeuA
c a 2-3Galβ1-4GlcNAcβl-3G
alβl-4G1cβ1-Car), ■-active ganglioside (Gal a l-3Galβl-4GlcNA
cβ1-6(NeuAc a 2-3Galβl-4G
lcNAcβl-3)Galβ1-401cNAcβl
-3Galβl-4Glcβ1-Car), GMlb(
NeuAca 2-3Galβl-3GalNAcβl
-4Galβl-4G1cβ1-Cer), C, Mla
(Galβ1-3GalNAcβ1-4(NeuAca
2-3) Galβl-401cβ1-Cer), 0M
2(GalNAcβl-4(NeuAca 2-3)C
alβ14G1cβ1-Car), GD2 (GalNA
cβl-4(NeuAca 2-8NeuAca 2-
3) Ga17? l-4Gtcβ1-Cer), GDla
(NeuAca 2-3)Galβl-3GalNAc
7? l-4(NeuAca 2-3)Galβl-4G
lcβ1-Cer), GDlb(Galβ1-3Ga
lNAcβ1-4(NauAc2-8NeuAca2
-3) Galβl-4Glcβ1-Car), GTlb
(NeuAca 2-3Galβl-3GalNAcβ
l-4 (NauAca 2-8NeuAca 2-3)
Galβl-4GIcβ1-Cer), 0M3 (Neu
Aca2-3Galβl-4Glcβ1-Car),
GD3 (NeuAca 2-8NeuAca 2-3G
alβl-40ICβ1-Cer), GT3(NeuA
ca 2-8NeuAca 2-8NeuAca 23
It does not react with Galβl-4GIcβ1-Cer).
（３）Ｎ−グリコリルノイラミン酸（ＮｅｕＧｃ）を含
む ＩＶコ（ＮｅｕＧｃ）ｎＬｃｌｃｅｒ（ＮｅｕＧｃ
ａ ２−３Ｃ：ａｌ β １−４Ｇ １ｃＮＡｃβ
］−３Ｇａｌβｌ−４Ｇｌｃ７？ １−Ｃｅｒ）、Ｖｌ
３（ＮｅｕＧｃ）ｎＬｃ５ｃｅｒ（ＮｅｕＧｃａ ２
−３Ｇａｌβｌ−４Ｇ１ｃＮＡｃβｌ−３Ｇａｌβ１−
４ＧＩｃＮＡｃβ１−３Ｇａｌβｌ−４Ｇｌｃβ１−Ｃ
ａｒ）、■−活性ガングリオシド（Ｇａｌ ａ １−３
Ｇａｌβｌ−４ＧｌｃＮＡｃβｌ−６（ＮｅｕＡｃ ａ
２−３Ｇａｌβｌ−４ＧｌｃＮＡｃβｌ−３）Ｇａｌ
βｌ−４Ｇ１ｃＮＡｃβｌ−３Ｇａｌβ１−４Ｇｌｃβ
１−Ｃａｒ）、Ｎ−グリコリルＧＭ３ガングリオシド（
ＮｅｕＧｃ ａ ２−３Ｇａｌβ１−４Ｇｌｃβ１−Ｃ
ｅｒ）とは反応しない。(3) IV co(NeuGc) nLclcer(NeuGc) containing N-glycolylneuraminic acid (NeuGc)
a 2-3C: al β 1-4G 1cNAcβ
]-3Galβl-4Glc7? 1-Cer), Vl
3(NeuGc)nLc5cer(NeuGca 2
-3Galβl-4G1cNAcβl-3Galβ1-
4GIcNAcβ1-3Galβ1-4Glcβ1-C
ar), ■-active ganglioside (Gal a 1-3
Galβl-4GlcNAcβl-6 (NeuAc a
2-3Galβl-4GlcNAcβl-3)Gal
βl-4G1cNAcβl-3Galβ1-4Glcβ
1-Car), N-glycolyl GM3 ganglioside (
NeuGc a 2-3Galβ1-4Glcβ1-C
er) does not react.
（４）シアル酸を含まない類縁糖脂質、ｎＬｃ４Ｃｅｒ
（ネオラクトテトラオシルセラミド、パラグロボシド、
Ｇａｌβｌ −４Ｇ １ｃＮＡｃβｌ −３Ｇａ １β
ｌ−４Ｇ１ｃβ１−Ｃｅｒ）、ペンタグリコジルセラミ
ド（Ｇａｌ ａ １−３Ｇａｌβｌ−４ＧｌｃＮＡｃβ
ｌ−３Ｇａｌβｌ−４Ｇｌｃβ１−Ｃｅｒ）、Ｌｃ４Ｃ
ｅｒ（ラクトテトラオシルセラミド、Ｇａｌβｌ−３Ｇ
１ｃＮＡｃβｌ−３Ｇａｌβ１−４Ｇｌｃβ１−Ｃｅｒ
）、アシアロＧＭ １ （Ｇａ ｌβｌ−３ＧａｌＮＡ
ｃβ１−４Ｇａｌβｌ−４Ｇｌｃβ１−Ｃｅｒ）、アミ
ノＣＴＨ（ＧｌｃＮＡｃβｌ−３Ｇａ １βｌ−４Ｇｌ
ｃβ１−Ｃｅｒ）、ラクトシルセラミド（Ｇ’ａｌβｌ
−４０１ｃβ１−Ｃａｒ）、Ｇｂ３Ｃｅｒ（グロポトリ
アオシルセラミド、Ｇａｌ ａ ｌ−４Ｇａｌβｌ−４
Ｇｌｃβ１−Ｃｅｒ）、７オルスマン（ＧａｌＮＡｃ
ａ ｌ−３ＧａｌＮＡｃβ１−３Ｇａｌ ａ ｌ−４Ｇ
ａｌβ１−４Ｇｌｃβ１−Ｃｅｒ）、Ｇｂ４Ｃｅｒ（グ
ロボシド、Ｇａ１ＮＡｃβ１３ＧａＬａ１−４Ｇａｌβ
ｌ−４Ｇ１ｃβ１−Ｃａｒ）、グルコシルセラミド（Ｇ
ｌｃβ１−Ｃａｒ）、ガラクトシルセラミド（Ｇａ １
β１−Ｃａｒ）、スルファチド（Ｈ５Ｏｘ−３Ｇａｌβ
１−Ｃｅｒ）とは反応しない。(4) Related glycolipids that do not contain sialic acid, nLc4Cer
(neolactotetraosylceramide, paragloboside,
Galβl -4G 1cNAcβl -3Ga 1β
1-4G1cβ1-Cer), pentaglycodylceramide (Gal a 1-3Galβ1-4GlcNAcβ
l-3Galβl-4Glcβ1-Cer), Lc4C
er (lactotetraosylceramide, Galβl-3G
1cNAcβl-3Galβ1-4Glcβ1-Cer
), asialo GM 1 (Galβl-3GalNA
cβ1-4Galβ1-4Glcβ1-Cer), amino CTH (GlcNAcβ1-3Ga 1β1-4Gl
cβ1-Cer), lactosylceramide (G'alβl
-401cβ1-Car), Gb3Cer (glopotriaosylceramide, Gal a l-4Galβl-4
Glcβ1-Cer), 7 orsman (GalNAc
a l-3GalNAcβ1-3Gal a l-4G
alβ1-4Glcβ1-Cer), Gb4Cer (globoside, Ga1NAcβ13GaLa1-4Galβ
l-4G1cβ1-Car), glucosylceramide (G
lcβ1-Car), galactosylceramide (Ga1
β1-Car), sulfatide (H5Ox-3Galβ
1-Cer).
（５）モノクローナル抗体（ＳＫ−８）の免疫グロブリ
ンクラスはＩｇＭであった。(5) The immunoglobulin class of the monoclonal antibody (SK-8) was IgM.
モノクローナル抗体（ＳＫ−８）を産生ずるハイブリド
ーマを樹立するための抗原、α２−３シアリルパラグロ
ボシドは、現在までのところ、化学合成した報告は見あ
たらず、試薬としての市販品も供給されていない。しか
し、これはヒト赤血球膜から単離することが出来る。す
なわち、ヒト赤血球膜総脂質をクロロホルム／メタノー
ル処理により抽出し、アルカリ処理により粗スフィンゴ
糖脂質画分を得、さらにＤＥＡＥ−セファデックス（フ
ァルマシア社製）カラムクロマトグラフィーによりモノ
シアロガングリオシド画分を得た後、ヤトロビーズ（ヤ
トロン社製）カラムクロマトグラフィーにより単離でき
る（Ｊ、 Ｂｉｏｌ、 Ｃｈｅｍ、、 ２６１．１７０
５７１７０６１（１９８６）、 Ｊ、 Ｂｉｏｌ、 Ｃ
ｈｅｍ、、 ２５３．８９６２−８９６７（１９７８）
）。To date, there have been no reports of chemical synthesis of α2-3 sialylparagloboside, an antigen for establishing hybridomas that produce monoclonal antibodies (SK-8), and no commercially available reagents have been found. . However, it can be isolated from human red blood cell membranes. That is, human red blood cell membrane total lipids were extracted by chloroform/methanol treatment, a crude glycosphingolipid fraction was obtained by alkali treatment, and a monosialoganglioside fraction was further obtained by DEAE-Sephadex (manufactured by Pharmacia) column chromatography. After that, it can be isolated by Yatrobeads (manufactured by Yatron) column chromatography (J, Biol, Chem, 261.170
5717061 (1986), J. Biol, C.
hem,, 253.8962-8967 (1978)
).
このようにして得た抗原となるガングリオシドをＷａｔ
ａｒａｉらの方法（Ｊ−Ｂｉｏｃｈｅｍ、、１０２．５
９−６７（１９８７））によりリポソームに埋め込む。The antigen ganglioside obtained in this way was
Arai et al.'s method (J-Biochem, 102.5
9-67 (1987)).
これをＥＬ Ｉ ＳＡ法、Ｌ Ｉ Ｌ Ａ（ｌｉｐｏｓ
ｏｍｅ ｉｍｍｕｎｅ １ｙｓｔｓａｓｓａｙ）法、限
界希釈法を使用して本願目的抗体を生産するセルライン
（）１イプリドーマ ＣＣ３３、微工研菌寄第１０４６
８号）を樹立した。This is called the EL I SA method, L I LA (lipos
The cell line that produces the target antibody of the present application using the ome immune 1st assay) method and the limiting dilution method ()1 Ipridoma CC33, Microtechnical Laboratory No. 1046
No. 8) was established.
ガングリオシドをリポソームに埋め込みそれを抗原とし
て免疫する今回のモノクローナル抗体作成法は、次のい
くつかの点で従来の方法より優れている。すなわち
（１）従来は、静脈内投与などが行われたが、簡単な腹
腔的投与で良い。This method of producing monoclonal antibodies, in which gangliosides are embedded in liposomes and used as antigens for immunization, is superior to conventional methods in several respects: That is, (1) conventionally, intravenous administration was performed, but simple intraperitoneal administration may be sufficient.
（２）免疫回数は、従来の方法は、数回であったが１回
で良い。(2) The number of immunizations may be one, whereas in the conventional method it was several times.
（３）免疫期間は、従来１〜数力月を要したが、投与後
３日間で良い。(3) The immunization period, which conventionally required one to several months, may be as long as three days after administration.
このようにして得たセルラインから常法によりモノクロ
ーナル抗体（ＳＫ−８）を得た。この抗体の性質は前記
のとおりである。A monoclonal antibody (SK-8) was obtained from the cell line thus obtained by a conventional method. The properties of this antibody are as described above.
（発明の効果）
本発明にかかわるモノクローナル抗体は、ＩＶ″（Ｎｅ
ｕＡｃ）ｎＬｃ４ｃｃｉｒおよびＩｔ／’（ＮｅｕＡｃ
）Ｌｃ４Ｃｅｒのいずれをも認識でき、しかもその他の
ガングリオシド類縁糖脂質とは反応しない高い特異性を
持つため、ＮｅｕＡｃ ａ ２−３Ｇａｌβｌ−４（３
）ＧｌｃＮＡｃを持つネオラクトテトラオシル系ガング
リオシドおよびラクトテトラオシル系ガングリオシド抗
原の超微量解析に極めて有用である。従って、これらの
抗原が発現している病体組織、細胞、体液、等における
抗原量を測定することにより、癌その他の疾病の診断が
可能となる。さらにこのモノクローナル抗体を用いた治
療もおこなえる。又［Ｖコ（ＮｅｕＡｃ）ｎＬｃ４ｃｅ
ｒは、インフルエンザウィルスが共通して認識する受容
体でもあり、広域インフルエンザワクチンとしても応用
可能である。(Effect of the invention) The monoclonal antibody according to the present invention is IV'' (Ne
uAc)nLc4ccir and It/'(NeuAc
)Lc4Cer and has high specificity that does not react with other ganglioside-related glycolipids.
) It is extremely useful for ultratrace analysis of neolactotetraosyl gangliosides and lactotetraosyl ganglioside antigens having GlcNAc. Therefore, by measuring the amount of antigens expressed in diseased tissues, cells, body fluids, etc., cancer and other diseases can be diagnosed. Furthermore, treatment using this monoclonal antibody can also be performed. Also [Vco (NeuAc)nLc4ce
r is also a receptor commonly recognized by influenza viruses, and can also be applied as a broad-spectrum influenza vaccine.
次に実施例により本発明を述べる。Next, the present invention will be described by way of examples.
実施例 １
ヒト赤血球から単離したα２−３シアリルバラグロボシ
ド（Ｊ、Ｂｉｏｌ、Ｃｈｅｍ、、２６１゜１７０５７−
１７０６１（１９８６）に基づき調製）を第１表に示す
処方により各成分を混入し、リポソームを作成した。Example 1 α2-3 sialylvala globoside isolated from human red blood cells (J, Biol, Chem, 261°17057-
17061 (1986)) was mixed with each component according to the formulation shown in Table 1 to prepare liposomes.
第１表
マウス−匹当りに用いたリポソーム作成処方σ２−３シ
アリルパラグロボシド ２１．６ ｎｍｏｌジパル
ミトイルホスファチジル ０．５ μｍｏｌコリン
コレステロール ０．５ μｍｏｌ
サルモネラ・ミネソタＲ５９５のＬＰＳ １０．０
μｇ（Ｓａｌｍｏｎｅｌｌａ ｍ１ｎｎｅｓｏｔａ）
（Ｓ、ｍ １ｎｅｓｏｔａに関しては、Ｇａ１ａｎｏｓ
、 Ｃ，ら、 Ｅｕｒ、 Ｊ。Table 1 Mouse - Liposome preparation recipe used per mouse σ2-3 Sialylparagloboside 21.6 nmol Dipalmitoyl phosphatidyl 0.5 μmol Choline cholesterol 0.5 μmol
Salmonella minnesota R595 LPS 10.0
μg (Salmonella m1nnesota)
(For S, m 1nesota, Ga1anos
, C. et al., Eur, J.
Ｂｉｏｃｈｅｍ、 ９．２４Ｅｒ２４９ （１９６９）
参照）これを３７℃で蒸発乾固後、クロロホルムで均一
にし、ロータリーエハホレーターでクロロホルムを乾固
してから、さらに真空ポンプで約−時間減圧した。これ
にＰＢＳを０．５ｍｇ加え、６０℃で５−６分保温して
乾固物をはがれ易くしてからポルテックスミキサーで充
分均一にすることによりリポソームを調製し、２２週令
のＢＡＬＢ／ｃマウスの腹腔内に投与した。Biochem, 9.24Er249 (1969)
(Reference) This was evaporated to dryness at 37°C, homogenized with chloroform, chloroform was dried to dryness using a rotary evaporator, and the pressure was further reduced for about an hour using a vacuum pump. Liposomes were prepared by adding 0.5 mg of PBS and incubating at 60°C for 5-6 minutes to make the dry substance easy to peel off, and then homogenizing it with a portex mixer to prepare liposomes. It was administered intraperitoneally to mice.
初回免疫後、３日目に牌蔵を摘出し、ミエローマ細胞（
Ｐ３−Ｘ６３−Ａｇ８−Ｕｌ）と３０％ＰＥＧ１００Ｏ
により細胞融合を行った。ＨＡＴ選択培地で培養後、出
現したハイブリドーマについてα２−３シアリルパラグ
ロボシドに対する抗体産生のスクリーニングをＥＬ Ｉ
ＳＡ法により行った。また特異性の検討はＥＬ Ｉ
ＳＡ法及びＬＩＬＡ法によった。これらによるアッセイ
の結果、特異抗体を産生じ、かつ反応性の高かったもの
について、限界希釈法によるクローニングを行った。On the 3rd day after the first immunization, the tiles were removed and myeloma cells (
P3-X63-Ag8-Ul) and 30% PEG100O
Cell fusion was performed by After culturing in HAT selection medium, the hybridomas that appeared were screened for antibody production against α2-3 sialylparagloboside using EL I.
This was done using the SA method. In addition, the study of specificity is carried out using EL I.
The SA method and LILA method were used. As a result of these assays, those that produced specific antibodies and had high reactivity were cloned by limiting dilution method.
このようにして最終的にσ２−３シアリルパラグロボシ
ドに対するモノクローナル抗体（ＳＫ−８）を産生ずる
ハイブリドーマ（ハイブリドーマ ＣＣ３３，ＦＥＲＭ
Ｐ−１０４６８として微工研に寄託されている）を
樹立した。In this way, a hybridoma (hybridoma CC33, FERM) that finally produces a monoclonal antibody (SK-8) against σ2-3 sialylparagloboside
P-10468, which has been deposited with the Institute of Fine Technology) was established.
実施例 ２
モノクローナル抗体（ＳＫ−８）のＩＶ’（ＮｅｕＡｃ
）ｎＬｃ４ｃｅｒおよび１ｖ３（ＮｅｕＡｃ）Ｌｃ４Ｃ
ｅｒに対する反応性を薄層クロマトグラフィー／酵素免
疫測定法において抗原量を変化させることにより示した
。（第１図）。Example 2 IV' (NeuAc) of monoclonal antibody (SK-8)
)nLc4cer and 1v3(NeuAc)Lc4C
Reactivity to er was demonstrated by varying the amount of antigen in thin layer chromatography/enzyme immunoassay. (Figure 1).
即ち、図に示した抗原量をシリカゲル薄層プレー ト
（Ｐｏｌｙｇｒａｍ Ｓｉｌ Ｇ、 Ｍａｃ
ｈｅｒｅｙ−Ｎａｇｅｌ、Ｆ、Ｒ，Ｇ、）上にスポット
し、クロロホルム／メタノール／水（５： ５ ： ｌ
、 ｖ／ｖ／ｖ）で展開した。プレートを乾燥後、１
％卵アルブミン−１％ポリビニルピロリドン溶液（Ａ液
）中にひたし、薄層表面をブロッキングすることにより
、非特異的吸着を阻止した。Ａ液を除去し、５Ｋ−８溶
液を加え、３０°０．２時間振盪保温後、Ｏ，１％Ｔｗ
ｅｅｎ２０のＰＢＳ溶液で洗浄、Ａ液中３７°ｃ、 ２
時間保温した。次いで、ペルオキシダーゼ結合二次抗体
（ウサギ抗マウスＩｇＧ）を加え、３７℃、２時間振盪
保温後、ＰＢＳで洗浄し、Ｈ，Ｏ，，４−クロロ−１−
ナフトール、トリス−ＨＣＱＩｌｋＷＩ液（ｐＨ７，２
）を含む基質溶液（Ｈｉｇａｓｈｉら、Ｊ、Ｂｉｏｃｈ
ｅｍ、、９５．１５１７−１５２０（１９８４）参照）
を加え、１０分後、蒸留水でプレートを洗浄、乾燥後、
スポットの発色を薄層クロマトグラムスキャナーで定量
した。That is, the amount of antigen shown in the figure was applied to a silica gel thin layer plate (Polygram Sil G, Mac
herey-Nagel, F, R, G,) and chloroform/methanol/water (5: 5: l
, v/v/v). After drying the plate, 1
% egg albumin-1% polyvinylpyrrolidone solution (liquid A) to block the surface of the thin layer to prevent non-specific adsorption. Remove A solution, add 5K-8 solution, keep shaking at 30° for 0.2 hours, and add O, 1% Tw.
Wash with een20 PBS solution, 37°C in solution A, 2
It was kept warm for hours. Next, peroxidase-conjugated secondary antibody (rabbit anti-mouse IgG) was added, and after incubation at 37°C for 2 hours with shaking, washing with PBS, H,O,,4-chloro-1-
Naphthol, Tris-HCQIlkWI solution (pH 7,2
) containing a substrate solution (Higashi et al., J. Bioch.
Em, 95.1517-1520 (1984))
After 10 minutes, wash the plate with distilled water and dry it.
The color development of the spots was quantified using a thin layer chromatogram scanner.
実施例 ３
モノクローナル抗体（ＳＫ−８）のＩＩ／”（ＮｅｕＡ
ｃ：）ｎＬｃ４Ｃｅｒ抗原に対する反応性をＬＩＬＡ法
において、抗体濃度を変化させることにより示した。こ
れにより、ｌ ” １００１００ｐの微量抗原が、定量
的に測定、解析できることが確かめられた。又、本抗体
は、ＩＶ３（ＮｅｕＡｃ）ｎＬｃ４Ｃｅｒ、 Ｉ’Ｖ’
（ＮｅｕＡｃ）Ｌｃ４Ｃｅｒ以外のガングリオシドや糖
脂質とは反応しないことが確かめられた（第２図）。Example 3 Monoclonal antibody (SK-8) II/” (NeuA
c:) Reactivity to nLc4Cer antigen was demonstrated by varying the antibody concentration in the LILA method. As a result, it was confirmed that a trace amount of l''100100p antigen can be quantitatively measured and analyzed.In addition, this antibody can be used for IV3(NeuAc)nLc4Cer, I'V'
It was confirmed that (NeuAc) does not react with gangliosides or glycolipids other than Lc4Cer (Figure 2).
即ち、マイグロブレートに図に示した希釈倍数の５Ｋ−
８抗体（２５μＱ）と種々の糖脂質及び蛍光マーカー（
０，ＩＭ力ルポキシフルオレスセイン）を含むリポソー
ム（シミリストイルホスファチジルコリン：コレステロ
ール：ジパルミトイルホスファチジン酸：糖脂質（０，
５μｍｏ１／Ｑ、５μｍｏ１１０．０５μｍｏｌ／Ｑ、
０５／Ｚｍｏｌ）懸濁液の１００倍希釈液（５μｌ）及
び２００倍希釈したモルモット血清を補体として加え（
２５μｌ）、３７℃、１時間、保温した。希釈はすべて
、０．１％ゼラチン−１０ｍＭベロナール−生理食塩水
緩衝液を加えることにより停止し、遊離した蛍光の強さ
を蛍光光時計により測定した（励起波長４９０ｒｏｎ
、測定波長５３０ｎｍ）抗体価は遊離した蛍光マーカー
％で表した。That is, 5K-
8 antibodies (25μQ) and various glycolipids and fluorescent markers (
Liposomes containing 0, IM (rupoxyfluorescein) (simyristoylphosphatidylcholine: cholesterol: dipalmitoylphosphatidic acid: glycolipids (0,
5μmol/Q, 5μmol110.05μmol/Q,
05/Zmol) suspension and 200-fold diluted guinea pig serum were added as complement (
25 μl) and kept warm at 37° C. for 1 hour. All dilutions were stopped by adding 0.1% gelatin-10mM veronal-saline buffer, and the intensity of the released fluorescence was measured by a fluorescence optical clock (excitation wavelength 490ron).
, measurement wavelength 530 nm) Antibody titer was expressed as % of free fluorescent marker.
試験に供されたガングリオシドは第２表に示される。The gangliosides tested are shown in Table 2.
第１図はモノクローナル抗体（ＳＫ−８）の抗原に対す
る反応性を薄層クロマトグラフィー／酵素免疫測定法に
より示したものである。
第２図はモノクローナル抗体の抗原に対する反応性をＬ
ＩＬＡ法により示したものである。
Ｏ−Ｏは第２表のＮｏｌの糖脂質を示し、・−・は第２
表のＮｏ２〜Ｎｏ２７の糖脂質を示す。
第１図
抗原量（ｐｍｏ立）FIG. 1 shows the reactivity of a monoclonal antibody (SK-8) to an antigen by thin layer chromatography/enzyme immunoassay. Figure 2 shows the reactivity of monoclonal antibodies to antigens.
This is shown using the ILA method. O-O indicates Nol glycolipid in Table 2, and ... indicates No. 2 glycolipid.
Glycolipids No. 2 to No. 27 in the table are shown. Figure 1 Antigen amount (pmo)
Claims (1)
シルセラミド及び￥Ｎ￥−アセチルノイラミニルラクト
テトラオシルセラミドの両者を特異的に認識できるモノ
クロナール抗体。 ２、請求項１記載のモノクローナル抗体を産生するハイ
ブリドーマ。[Scope of Claims] 1. A monoclonal antibody that can specifically recognize both \N\-acetylneuraminyllactotetraosylceramide and \N\-acetylneuraminyllactotetraosylceramide. 2. A hybridoma producing the monoclonal antibody according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1027507A JPH02207798A (en) | 1989-02-08 | 1989-02-08 | Monoclonal antibody to ganglioside and hybridoma capable of producing antibody thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1027507A JPH02207798A (en) | 1989-02-08 | 1989-02-08 | Monoclonal antibody to ganglioside and hybridoma capable of producing antibody thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02207798A true JPH02207798A (en) | 1990-08-17 |
Family
ID=12223051
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1027507A Pending JPH02207798A (en) | 1989-02-08 | 1989-02-08 | Monoclonal antibody to ganglioside and hybridoma capable of producing antibody thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02207798A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005517901A (en) * | 2001-12-21 | 2005-06-16 | ビスカム・アーゲー | Method for determining individual responsiveness to mistletoe lectin |
-
1989
- 1989-02-08 JP JP1027507A patent/JPH02207798A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005517901A (en) * | 2001-12-21 | 2005-06-16 | ビスカム・アーゲー | Method for determining individual responsiveness to mistletoe lectin |
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