JPH0398596A - Monoclonal antibody - Google Patents
Monoclonal antibodyInfo
- Publication number
- JPH0398596A JPH0398596A JP23508589A JP23508589A JPH0398596A JP H0398596 A JPH0398596 A JP H0398596A JP 23508589 A JP23508589 A JP 23508589A JP 23508589 A JP23508589 A JP 23508589A JP H0398596 A JPH0398596 A JP H0398596A
- Authority
- JP
- Japan
- Prior art keywords
- strain
- iia
- ppi
- antibody
- amino acid
- Prior art date
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Landscapes
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Abstract
Description
【発明の詳細な説明】
［Ｉ１！業上の利用分野〕
本発明は、モノクローナル抗体に関する．更に詳しくは
、ポテトセリンプロテアーゼインヒビターの分子構造と
機能の研究に有用なモノクローナル抗体に関する．
［従来の技術とその問題点］
一般に、モノクローナル抗体は、各種の抗原抗体反応の
測定に利用され、特定の蛋白貢等の抗原抗体反応の機構
を検討するために有用であることは周知である．
しかし、特定の抗原抗体反応の機構の検討に有用なモノ
クローナル抗体は、該反応に対する抗体として機能しな
ければならないので、かかる抗体は、特定の蛋白質等の
目的とする抗原抗体反応に適合するように遺伝子工学の
方法等を用いて個々に生合戊しなければならない．そし
て該生合成の手段も個々に異る．
本発明者等は、ポテトセリンプロテアーゼインヒビター
（ＰＰＩ−ＩＩａ）の分子構造と機能の研究を行ってい
るが、この研究に抗原抗体反応を利用することが、該イ
ンヒビターの高次構造を解明する上で必要と考えられた
．
そこで、体内免疫法で免疫したＢＡＬＢ／ＣマウスのＭ
細胞と主エローマ細胞（Ｐ３Ｕ１）を５：１の比で融合
させて得られたパイプリドーマから、特異抗体産生細胞
をスクリーニングすることにより、２種類の作成抗体１
−８０ （ ＩｇＧ＋）と１−１１＾（ＩｇＭ）を得た
．そして、該抗体がいづれも， ＰＰＴ−ＩＩａから調
製された＾ｃｔｌｖｅ Ｆｒａｇｍｅｎｔと抗原抗体反
応をする一方、還元ＣＭ化および還元アセトアよド化さ
れたＰＰＩ−ＩＩａとは反応しないことを知見して、本
発明を完成した．
抗原抗体反応についての以上の事実は、＾ｅｔＩｖｅＦ
ｒａｇ嘗ｅｎｔ内に存在する抗原決定部位にとって鎮内
Ｓ−Ｓ結合によって保持されている高次構造が１１Ｊ！
であることを示唆するものである．以上の記述から明ら
かなように、本発明の目的は、ポテトセリンインブロテ
アーゼヒビターの研究に有用なモノクローナル抗体およ
びその製造法を提供することである．
［問題点を解決するための手段］
本発明は、下記（１）の構成を有する．（１）ポテトセ
リンプロテアーゼインヒビターＩＩ ａ （ＰＰＩ−
ＩＩ ａ）で免疫しｋマウス８＾ＬＢ／Ｃの牌細胞とマ
ウスミローマ細ＱＰ３Ｕｌをポリエチレングリコール４
０００を用いて細胞融合させ、該融合物をＰＰＩ−Ｈａ
に対する特異抗体産生ハイブリドーマでスクリーニング
し、および限界希釈法によるクローニングを行って １
−１１Ａ株および１−８Ｄ株を取得し、該１−１１＾株
に産生せしめてなるモノクローナル抗体ＬｇＭおよび該
１−６Ｄ株に産生せしめてなるモノクローナル抗体１３
Ｇ．，
本発明の構成と効果につき以下に詳述する．ｌ．モノク
ローナル抗体の作製
■ポテトセリンプロテアーゼインヒビターＩＩ ａ（Ｐ
ＰＩ−１１ ａ）によるＢＡＬＢ／Ｃマウスの免疫：Ｐ
ＰＩｉｌａ抗原溶液（ＳＯＯμｇ／ ＩＪＬｏｆ Ｄｕ
ｌｂｅｃｃｏ−ＰＢＳ） ２００μｌｌとＦｒｅｕｎｄ
’ｓ Ｃｏｍｐｌｅｔｅ ＡＪｕｖａｎｔ ２００μぶ
を混合乳化させ、マウス（ ＢＡＬＢ／Ｃ．雌．８週令
）の腹腔内に注射した．
２週間後、同じ操作で追加免疫を行った．さらに２週間
後に １００μ角の抗原溶液のみを腹腔内に注射した．
その３日後に牌臓を摘出して牌細胞を調製した．
■牌ｍ胞とマウス由来ミエローマｍ胞とのｍ胞融合；
対数増殖期にあるマウスミエローマｍ胞（Ｐ３Ｕ１）２
ｘ １０’ ｃｅｌｌｓと上記調製牌細胞１０Ｘ １
０’ ｃｅｌｌｇ−細胞比ｌ／５−を４７％Ｐｏｌｙｅ
ｔｈｙｌｅｒ＋ｅｇｌｙｃｏｌ ４０００４．７％ ０
１ｍｅｔｈｙｌｓｕｌｆｏｘｌｄｅ／ｒｓｃｏｖｅ Ｍ
ｏｄｌｆｌｄｅＤｕｌｂｅｃｃｏ Ｍｅｄｌｕ＋ｓを用
いて融合させた．■ハイブリドーマの培養と特異抗体産
生ハイブリドーマのクローニング：
融合細胞を｝ＩＡＴ−ＩＭＤＭ （Ｈｙｐｏｘａｎｔｈ
Ｉｎｅ−ａｍｌｍｏｐｔａｒＩｎ−Ｊｈｙｍｆｄｌｎｅ
−Ｉｓｃｏｖｅ ＭｏｄｌｆＩｅｄ Ｄｕｌｂｅｃ
ｃｏ Ｍｅｄ１ｕｍ（ ａｍｌｎｏｐｔｅｒｌｎを除
外した培地）で４〜５日間培養した．培養上清について
ＥＬｒＳＡ法により抗体陽性細胞を選択し、限界希釈
法によるクローニングを行った．
この結果をまとめて表１、表２に示す．表１ 細胞融合
および抗体産生パイブリドーマの培養
−１０％ＦＣＳ）で７日間培養し，さらにＨＴ−ＩＭＤ
ｋｌ表２．抗体産生パイプリドーマのクローニング５−
８Ａ株よりクローニングされた抗体産生株１１１＾５−
ＩＦ株よりクローニングされた抗体産生株１−６Ｄ（
）特異抗体産生ウエル数
注）複数コロニー生成ウエルは
存在しなかった．
＋１ ．作製モノクローナル抗体の性質■モノクローナ
ル抗体の調製：
ポテトセリンプロテアーゼインヒビターＩＩ ａに対す
る抗体を産生ずるモノクローンハイブリドーｖ （１
−６Ｄ株，１−１１＾株）を！０％ＦＣＳ−ＩＭＤＭで
培養して上清を得た．ついで、硫安１／２飽和で抗体を
分別沈殿させ、以下実験に供した．
■モノクローナル抗体のクラス、サブクラスの同定：
ウサギー抗マウスＩｇＧ＋． ＩｇＧ２ａ， ＩｇＧｚ
ｂ． ＩｇＧｓ（ＺＹＭＥＤ社）およびヤギー抗マウス
ＩｇＭ ＣＭＩＬＥＳ社）を用い、 ＥＬＩＳＡ法によ
り、上記２株の産生ずるモノクローナル抗体の同定を行
った．その結果第１図に示すように， ！−８Ｄ株と
１−１１Ａ株の彦生ずる抗体は各々ＩｇＧ．とＩｇＭと
同定された．
■モノクローナル抗体の抗原特異性：
１−６０株と １−１１＾株の彦生ずる抗体の力価を評
価したところ、１−６０株の抗体は、第２図に示すよう
に約２５５０倍希釈上清で、抗体飽和率６０％を与える
ことが明らかになった．これに対し、１−１１Ａ株の抗
体は２５０倍希釈で同じ６０％の値を与えた．従って力
価の高かった１−６Ｄ株の産生ずる抗体について、以下
の実験を行うことにした．
＋１１．−Ｅ−ノクローナル抗体（１−６Ｄ）に対する
ＰＰＩ−■ａの抗原決定部位
■Ｉ−６Ｄ株の産生ずるモノクローナル抗体（１−６０
−Ｍａｂ）に対する＾ｃｔｌｖｅ Ｆｒａｇｍｅｎｔの
抗原特異性：ＰＰＩ−ＩＩａの抗原決定部位に関与する
アミノ酸残基を含むベプチドを分離する前段階として，
ＰＰＩ−ＩＩａ（構成アミノ酸残基数９７）より調製
したインヒビター活性をもつ＾ｃｔｉｖｅ Ｆｒａｇｍ
ｅｎｔ（構成アミノ酸残基数４５）の抗原特異性を検討
し、第３図に示す結果を得た．
＾ｃｔｉｖｓ Ｆｒａｇｍｅｎｔ（ア≧ノ酸配列は後述
）が、ＰＰＩ−ＩＩａと１−６Ｄ−Ｍａｂの抗原抗体反
応を完全に阻害したことから、このＦｒａｇ璽ｅｎｔに
抗原決定部位が存在することが明示された．
■抗原決定部位を構成するアミノ酸残基を含むベブチド
の分離絹製：
ＰＰＴ−Ｈａは分子内に６ヶのジスルフィド結合を有し
ているので、多数のループ断片から構成されていると考
えられる．従って、ループ断片そのものを分子より分割
させ、抗原活性を示すループ断片を分離精製することを
試みた．
（ａ） ＰＰＩ−Ｔｌ ａのジスルフィド結合の還元開
裂とこれに次ぐ、２−Ｎｉｔｒｏ−Ｓ−ｔｈｌｏｃｙａ
ｎｏｂａｎｘｏａｔｅ （ＮＴＣＢ）によるシスティン
残基のアミノ基側ベブチド結合の切断
ＰＰＩ−ＩＩａを６Ｎ塩酸グアニジン−０．２Ｍ Ｔｒ
ｉｓ−ＨＣＩ，ｐＨ ８．０中でジチオスレイトール処
理することにより、分子内ジスルフィド結合を還元開裂
させた．次いで、生じたシスティン残基をＮＴＣ８で修
飾するとともに、そのアミノ基側のベブチド結合を切断
した．ついで反応混液を５０％酢酸を用いたＳｅｐｈａ
ｄｅｘ Ｇ−１ｓカラムによるゲル濾過に供し、過剰の
試薬を除去した．ペブチド画分を次の実験に供した．
（ｂ）逆相系カラムを用いた高速液体クロマトグラフィ
ー（ＨＰＬＣ）によるベブチドの分離精製上記ベブチド
画分をＣｌａ−７−００５−｝１カラム（４．６ｘ ２
ＳＯＩＩｓ）を用いたＨＰＬＣに供与し、第４図に示す
溶液パターンを得た．
なお、分離されたＮＴＣＢ−１〜ＮＴＣＢ−７の各ピー
クの抗原活性は、各々のピークに含まれるペプチドを固
相に吸着させたのち、１−６０−Ｍａｂを結合させＥＬ
ＩＳＡ法によって測定した．二次抗体に結合させたＨｏ
ｒｓｅｒａｄｌｓｈ − Ｐｅｒｏｘｌｄａｓｅの酵素
活性の測定値を第４図中の各溶出ピーク名の後に記した
．同じ条件で測定したＰＰＩ−ＩＩａおよびＮＴＣＢ処
理ＰＰＩｉｌａ分割混合ペプチドの活性は、それぞれＯ
、４３と０．１３であった．
■抗原活性を有すベブチドのアミノ酸組成：抗原活性の
高かったＮ丁ＣＢ−３， ＮＴＣＢ−４， ＮＴＣＢ−
５（いづれも第４図中に陰をほどこしたピーク）につい
て、そのアミノ酸組成分析を行った．その結果を第３表
に、ＰＰＩＩＩａのアくノ酸配列上相同な部位のアミノ
酸組成とともに示す．
第３表 ＨＰＬＣ生成ベブチドのアミノ酸組成注、＊（
）内数は理論値を示す．
＋１７ＣＢ−３とＮ丁ＣＢ−４のアミノ酸組成はＰＰＩ
−ＩＩｇのアミノ酸配列（第５図）上の５７−９７に近
似していた．またＮＴＣＢ−５は、反応部位Ｌｙｓ”−
Ｓｅｒ”を含む５７−８７に一致していた．
以上の結果、１−６０−Ｍａｂによって認識される抗原
決定部位を構成する主要アミノ酸残基は、セリンブロテ
アーゼに対する反応部位（第５図中Ｏ印で囲んだ−Ｌｙ
ｓ−Ｓｅｒ）を含んだ（ｙｓ８７〜（ｙｓ８６の間のル
ープに存在することが明らかになった．■抗原決定部位
構成主要アミノ酸残基がＰＰＩ−１ｆａの−（Ｈｙｓ８
７〜Ｃｙｓ■一間のループに存在していることの間接的
証拠：
ＨＰＬＣで分踵されたＮ丁ＣＢ−５ベプチドに抗原活性
が認められたことから、−（ｙｓ！７〜Ｃｙｓ６８−の
間のループに抗原決定部位を構成する主要なアミノ酸残
基が存在していることが直接証明された．ここでは，
ＰＰＴ−■ａとアミノ酸配列において相同性の高いＰＰ
Ｉ−１１ｂの抗原活性を比較することによって、抗原決
定部位を構成しているアミノ酸残基を明らかにすること
を試みた．
その結果第６図に示すように、固相吸着ＰＰ■−ＩＩ
ｍと１−６０−Ｍａｂの抗原抗体反応に対するＰＰＩ−
１１ｂによる拮抗阻害は全く認められなかった．このこ
とからアミノ酸配列上相同性が高いにも拘らず、ＰＰＩ
−１１ｂには、このモノクローナル抗体に対する抗原決
定部位は存在しないことが明らかになった．
１−６Ｄ−ＭａｂはＰＰＩ−ＩＩａの＾ｃｔｉｖａ Ｆ
ｒａｇｍｅｎｔを抗原として認識し、また＾ｃｔｌｖｅ
Ｆｒａｇｍｅｎｔ分子由来のＮＴＣＢ−５ベブチドを
も認識することは既に記述したとおりである．いま、ア
ミノ酸配列が決定されているＰＰＩ−ＩＩａの＾ｃｔｌ
ｖａ Ｆｒａｇ＋ａｅｎｔとＰＰＩ−１１ｂの＾ｃｔｌ
ｖｅ Ｆｒａｇｍ＠ｎｔ（共に第７図に示す）を比較す
ると、ＰＰＩ−ＩＩａ＾．Ｆ．に抗原活性があり、ＰＰ
Ｉ−１１ｂ＾，Ｆ．に抗原活性がなかったことから、０
で囲んだ相同のア暑ノ酸残基は抗原決定部位に関与して
いす、ロで囲んだ相異アミノｍ残基のみが関与している
ことが推測された．また、一Ｃｙｓｓ７〜Ｃｙｓ＠Ｂ一
のループに限って、その相同性を比較すると下に示すよ
うに、反応部位を含む−Ｔｙｒ−Ｌｙｓ−Ｓｅｒ−は相
同であることから、ＰＰＩ−ＩＩａ＾．Ｆ，のアミノ酸
配列のうち、＾ｓｐ”−Ｓｅｔ” （Ｈｅｘａｐｅｐｔ
ｌｄｅ）のアミノ酸側鎮が、抗原一抗体反応にとってＩ
ｉ要であることが示唆された．
Ｒｅａｃｔｉｖｅ ｓｉｔｅ
＾
［効果］
・ポテトセリンプロテアーゼインヒビターＩＩ ａ（Ｐ
ＰＩ−ＩＩａ）で免疫したマウスＢＡＬＢ／Ｃの牌細胞
とマウスミエローマ細胞Ｐ３１１１を Ｐｏｌｙｅｔｈ
ｙｌｅｎｅｇｌｙｃｏ１ ４０００を用いて細胞融合さ
せた．ついで、ＰＰＩ−ＩＩａに対する特異抗体産生八
イプリドーマのスクリーニングと限界希釈法によるクロ
ーニングを行い、 １−１１Ａ株と１−６Ｄ株を得た．
１−１１Ａ株および１−６Ｄ株の産生ずるモノクローナ
ル抗体は、各々 ＩｇＭとＩｇＧ，であった．また、１
−６０株の培養上清の抗体力値は１−１１＾の約ｌθ倍
であった．
・１−８０株の産生ずるモノクローナル抗体（ １−６
Ｄ−Ｍａｂ）は、ＰＰＩ−ＩＩａ （構成アミノ酸残
基９７）とＰＰＩ−ＩＩａ由来の＾ｂｔｌｖｅ Ｆｒａ
ｇｍｅｎｔ （構成アミノ残基４５）と共に認識し抗
原一抗体反応した．このことから抗原決定部位は＾ｂｔ
１ｖａ Ｆｒａｇｍｅｎｔ内に存在することが示された
．
・抗原活性を保持するベブイチドを得るため、ＰＰＩ−
ＩＩａのジスルフィド結合を還元開裂させたのち、２−
ｎＩｔｒｏ−Ｓｔｈｌｏｃｙａｎｏｂｅｎｘｏａｔｅ
（ＮＴＩＩ：Ｂ）によりシステイン残基のアミノ基側の
ベブチド結合を切断した．ついで、Ｃ，，−７−００５
−｝１カラムを用いた逆相ＨＰＬＣにより、抗原活性を
保持した３つのべブチド（ ＮＴＣＢ−３、ＮＴＣＢ−
４、ＮＴＣＢ−５ ）を分離した．・分離したべブチド
のうちＮＴＣＢ−５は、そのアミノ酸組戒から、＾ｂｔ
ｉｖｅ Ｆｒａｇｍｅｎｔ （Ｓｅｒｓ４−＾ｒ１８
）内のＣｙｓ”−Ｖａｌ”に相当するベブチドであるこ
とが明らかとなった． ＮＴＣＢ−３とＮＴＣＢ−４は
互にア主ノ酸組成が近似していて、いづれもＰＰＩ−Ｉ
Ｉａ （＾ｌａ’−＾１ａ９？）のうち、ＮＴＣＢ−５
ベブチドも含んだベブチドＣｙｓ”−八１ａ４？である
ことが推定された．後者の２つのべブチドは、ＮＴＣＢ
による切断が不充分な結果生じたべブチドであることが
推測された．・ＰＰＩ−１１ ａ由来の＾ｂｔ１ｖｅ
Ｆｒａｇｍｅｎｔ （抗原活性有り）とＰＰＩ−１１
ｂ由来の＾ｂｔｌｖｅ Ｆｒａｇｍｅｎｔ （抗原活
性無し）のアミノ酸配列の比較から、−（ｙＳＳ？〜Ｃ
ｙｓ１１Ｍ一間のループを形成しているアミノ酸残基の
うち、特に＾ｐｓ”−Ｔｈｒ−＾ｓｎ−ＩＩｓ−＾１ａ
−Ｓａｒ”のＨｅｘａｐｅｐｔｌｄａの側鏡が抗原決定
部位の構成に主要であることが示唆された．[Detailed Description of the Invention] [I1! Field of Industrial Use] The present invention relates to monoclonal antibodies. More specifically, it relates to a monoclonal antibody useful for studying the molecular structure and function of potato serine protease inhibitor. [Prior art and its problems] Monoclonal antibodies are generally used to measure various antigen-antibody reactions, and it is well known that they are useful for studying the mechanism of antigen-antibody reactions such as those caused by specific proteins. ．． However, monoclonal antibodies useful for studying the mechanism of a specific antigen-antibody reaction must function as antibodies against that reaction, so such antibodies must be compatible with the target antigen-antibody reaction of a specific protein, etc. They must be synthesized individually using methods such as genetic engineering. The means of biosynthesis also differ among individuals. The present inventors are conducting research on the molecular structure and function of potato serine protease inhibitor (PPI-IIa), and the use of antigen-antibody reactions in this research will help elucidate the higher-order structure of the inhibitor. It was considered necessary. Therefore, the M
By screening for specific antibody-producing cells from pipulidoma obtained by fusing cells and main eloma cells (P3U1) at a ratio of 5:1, two types of antibodies 1 were generated.
-80 (IgG+) and 1-11^ (IgM) were obtained. Then, they discovered that while all of these antibodies had an antigen-antibody reaction with the ^ctlve fragment prepared from PPT-IIa, they did not react with PPI-IIa that had been reduced to CM and reduced to acetate. The invention has been completed. The above facts about antigen-antibody reactions are
For the antigen-determining site present in the rag ent, the higher-order structure maintained by the internal S-S bond is 11J!
This suggests that. As is clear from the above description, an object of the present invention is to provide a monoclonal antibody useful for research on potato serine inbrotease inhibitors and a method for producing the same. [Means for solving the problems] The present invention has the following configuration (1). (1) Potato serine protease inhibitor II a (PPI-
II a) immunized with mouse 8^LB/C tile cells and mouse myoma fine QP3U1 in polyethylene glycol 4.
000 for cell fusion, and the fusion was transformed into PPI-Ha
1 by screening with hybridomas producing specific antibodies against the target and cloning by limiting dilution method.
-11A strain and 1-8D strain were obtained, and monoclonal antibody LgM produced by the 1-11^ strain and monoclonal antibody 13 produced by the 1-6D strain
G. The structure and effects of the present invention are detailed below. l. Preparation of monoclonal antibody ■ Potato serine protease inhibitor II a (P
Immunization of BALB/C mice with PI-11 a): P
PIila antigen solution (SOOμg/IJLof Du
lbecco-PBS) 200 μl and Freund
200 μl of ''s Complete AJuvant was mixed and emulsified and injected intraperitoneally into mice (BALB/C, female, 8 weeks old). Two weeks later, a booster immunization was performed using the same procedure. Two weeks later, a 100μ square antigen solution was injected intraperitoneally.
Three days later, the spleen was removed and spleen cells were prepared. ■M-cell fusion of tile M-cell and mouse-derived myeloma M-cell; Mouse myeloma M-cell (P3U1) 2 in logarithmic growth phase
x 10' cells and the above prepared tile cells 10x 1
0' cellg-cell ratio l/5-47%Polye
tyler+eglycol 40004.7% 0
1methylsulfoxlde/rscove M
The fusion was performed using Dulbecco Medlu+s. ■Culture of hybridomas and cloning of specific antibody-producing hybridomas: IAT-IMDM (Hypoxanth
Ine-amlmoptarIn-Jhymfdlne
-Iscove ModlfIed Dulbec
The cells were cultured in coMed1um (medium excluding amlnopterln) for 4 to 5 days. Regarding the culture supernatant, antibody-positive cells were selected by the ELrSA method and cloned by the limiting dilution method. The results are summarized in Tables 1 and 2. Table 1 Culture of cell fusion and antibody-producing hybridomas (10% FCS) for 7 days, and further HT-IMD
kl table 2. Cloning of antibody-producing piglidoma 5-
Antibody-producing strain 111^5- cloned from 8A strain
Antibody-producing strain 1-6D cloned from IF strain (
) Number of wells producing specific antibodies Note) There were no wells producing multiple colonies. +1. Properties of monoclonal antibodies produced ■ Preparation of monoclonal antibodies: Monoclonal hybrid v (1) that produces antibodies against potato serine protease inhibitor II a
-6D stock, 1-11^ stock)! The supernatant was obtained by culturing in 0% FCS-IMDM. The antibody was then precipitated fractionally with 1/2 saturation of ammonium sulfate and used in the following experiments. ■Identification of monoclonal antibody class and subclass: Rabbit anti-mouse IgG+. IgG2a, IgGz
b. The monoclonal antibodies produced by the above two strains were identified by ELISA using IgGs (ZYMED) and goat anti-mouse IgM (CMILES). As shown in Figure 1, the result is ! -8D strain and
The antibodies produced by Hikou 1-11A strain are each IgG. It was identified as IgM. ■Antigen specificity of monoclonal antibodies: When we evaluated the titer of Hiko-generated antibodies for the 1-60 strain and 1-11^ strain, we found that the antibody for the 1-60 strain was diluted approximately 2550-fold as shown in Figure 2. It was revealed that the antibody saturation rate was 60%. In contrast, the antibody of the 1-11A strain gave the same value of 60% at 250-fold dilution. Therefore, we decided to conduct the following experiment using the antibody produced by the 1-6D strain, which had a high titer. +11. -Antigen-determining site of PPI-a against E-noclonal antibody (1-6D) - Monoclonal antibody (1-60 produced by I-6D strain)
Antigen specificity of ^ctlve Fragment for -Mab): As a preliminary step to isolate peptides containing amino acid residues involved in the antigen-determining site of PPI-IIa,
Active Fragm with inhibitor activity prepared from PPI-IIa (number of constituent amino acid residues: 97)
The antigen specificity of ENT (constituting 45 amino acid residues) was investigated, and the results shown in FIG. 3 were obtained. Since the ^ctivs Fragment (the amino acid sequence will be described later) completely inhibited the antigen-antibody reaction between PPI-IIa and 1-6D-Mab, it was clearly demonstrated that an antigen-determining site exists in this Fragent. Ta. ■ Separation of bebutide containing amino acid residues constituting the antigen-determining site made of silk: PPT-Ha has six disulfide bonds in its molecule, so it is thought to be composed of many loop fragments. Therefore, we attempted to separate and purify loop fragments that exhibit antigenic activity by dividing the loop fragments themselves from molecules. (a) Reductive cleavage of the disulfide bond of PPI-Tla followed by 2-Nitro-S-thlocya
Cleavage of the bebutide bond on the amino group side of the cysteine residue using nobanxoate (NTCB)
The intramolecular disulfide bond was reductively cleaved by treatment with dithiothreitol in is-HCI, pH 8.0. Next, the resulting cysteine residue was modified with NTC8, and the bebutide bond on the amino group side was cleaved. The reaction mixture was then purified with Sepha using 50% acetic acid.
The sample was subjected to gel filtration using a dex G-1s column to remove excess reagent. The peptide fraction was used in the following experiment. (b) Separation and purification of bebutide by high performance liquid chromatography (HPLC) using a reverse phase column.
SOIIs) was applied to HPLC to obtain the solution pattern shown in Figure 4. The antigen activity of each peak of separated NTCB-1 to NTCB-7 can be determined by adsorbing the peptides contained in each peak onto a solid phase, then binding 1-60-Mab to EL.
Measured by ISA method. Ho conjugated to secondary antibody
Measured values of the enzymatic activity of rseradlsh-Peroxldase are shown after each elution peak name in FIG. The activities of PPI-IIa and NTCB-treated PPIila split mixed peptides measured under the same conditions were respectively O
, 43 and 0.13. ■Amino acid composition of bebutide with antigenic activity: NchoCB-3, NTCB-4, and NTCB- had high antigenic activity.
5 (all peaks shaded in Figure 4) were analyzed for their amino acid composition. The results are shown in Table 3 along with the amino acid composition of homologous sites in the acetonic acid sequence of PPIIIa. Table 3 Amino acid composition of HPLC-generated bebutide Note, *(
) The numbers in ) indicate theoretical values. The amino acid composition of +17CB-3 and NchoCB-4 is PPI
It was similar to 57-97 in the amino acid sequence of -IIg (Figure 5). In addition, NTCB-5 has a reactive site Lys”-
57-87 containing "Ser". As a result, the main amino acid residues constituting the antigen-determining site recognized by 1-60-Mab are the reactive site for serine protease (O -Ly circled with a mark
It was revealed that the main amino acid residues constituting the antigen-determining site are present in the loop between (ys87 and (ys86) containing -(Hys8) of PPI-1fa.
Indirect evidence that CB-5 peptide exists in the loop between -(ys!7 and Cys68-): Antigenic activity was observed in the HPLC separated N-Cys CB-5 peptide. It was directly demonstrated that the main amino acid residues constituting the antigen-determining site are present in the intervening loop.
PP with high homology in amino acid sequence to PPT-■a
By comparing the antigenic activities of I-11b, we attempted to clarify the amino acid residues that constitute the antigen-determining site. As a result, as shown in Fig. 6, solid phase adsorption PP■-II
PPI- on the antigen-antibody reaction of m and 1-60-Mab
No competitive inhibition by 11b was observed. This shows that despite the high amino acid sequence homology, PPI
It was revealed that -11b does not have an antigen-determining site for this monoclonal antibody. 1-6D-Mab is PPI-IIa ^ctiva F
ragment as an antigen, and also ^ctlve
As already described, it also recognizes NTCB-5 bebutide derived from Fragment molecules. The amino acid sequence of PPI-IIa has now been determined.
va Frag+aent and PPI-11b ^ctl
ve Fragm@nt (both shown in FIG. 7), PPI-IIa^. F. has antigenic activity and PP
I-11b^, F. Since there was no antigenic activity in
It was inferred that the homologous amino acid residues surrounded by squares are involved in the antigen-determining site, and only the different amino acid residues circled in squares are involved. In addition, when comparing the homology of only one Cyss7 to Cys@B loop, as shown below, the -Tyr-Lys-Ser- containing the reaction site is homologous, so PPI-IIa^. Among the amino acid sequences of F, ^sp”-Set” (Hexapept
The amino acid flanker of Ide) is important for the antigen-antibody reaction.
It was suggested that this is important. Reactive site ＾ [Effect] ・Potato serine protease inhibitor II a (P
Polyeth
Cell fusion was performed using ylene glycol 4000. Subsequently, eight pridomas producing specific antibodies against PPI-IIa were screened and cloned by limiting dilution method to obtain strains 1-11A and 1-6D.
The monoclonal antibodies produced by the 1-11A strain and 1-6D strain were IgM and IgG, respectively. Also, 1
The antibody titer value of the culture supernatant of strain -60 was approximately lθ times that of 1-11^.・Monoclonal antibody produced by strain 1-80 (1-6
D-Mab) consists of PPI-IIa (constituent amino acid residue 97) and PPI-IIa-derived^btlve Fra
gment (constituent amino acid residue 45) and caused an antigen-antibody reaction. From this, the antigen-determining site is ^bt
1va Fragment.・To obtain Bebuitide that retains antigenic activity, PPI-
After reductive cleavage of the disulfide bond of IIa, 2-
nItro-Sthlocyanobenxoate
(NTII:B) was used to cleave the bebutide bond on the amino group side of the cysteine residue. Then, C,, -7-005
-}1 column, three peptides (NTCB-3, NTCB-
4, NTCB-5) was isolated.・Among the isolated bebutides, NTCB-5 is ^bt due to its amino acid composition.
ive Fragment (Sers4-^r18
) was found to be a bebutide corresponding to Cys"-Val". NTCB-3 and NTCB-4 have similar main amino acid compositions, and both are PPI-I.
Of Ia (^la'-^1a9?), NTCB-5
The latter two bebutides were estimated to be Cys”-81a4?, which also contains bebutide.
It was speculated that the bebutide was generated as a result of insufficient cleavage by・^bt1ve derived from PPI-11a
Fragment (with antigenic activity) and PPI-11
From the comparison of the amino acid sequences of the ^btlve Fragment (no antigenic activity) derived from B, -(ySS?~C
Among the amino acid residues forming the loop between ys11M, especially ^ps”-Thr-^sn-IIs-^1a
It was suggested that the Hexapeptlda side mirror of "-Sar" is important in the configuration of the antigen-determining site.
第１〜７図は、本発明のモノクローナル抗体の調製工程
または物性を説明するための説明図である．1 to 7 are explanatory diagrams for explaining the preparation process and physical properties of the monoclonal antibody of the present invention.
Claims (1)
Ｐ I −IIａ）で免疫したマウスＢＡＬＢ／Ｃの■細胞
とマウスミローマ細胞Ｐ３Ｕ１をポリエチレングリコー
ル４０００を用いて細胞融合させ、該融合物をＰＰ I
−IIａに対する特異抗体産生ハイブリドーマでスクリー
ニングし、および限界希釈法によるクローニングを行っ
て I −１１Ａ株および I −６Ｄ株を取得し、該 I −
１１Ａ株に産生せしめてなるモノクローナル抗体１ｇＭ
および該 I −６Ｄ株に産生せしめてなるモノクローナ
ル抗体１ｇＧ＿１。(1) Potato serine protease inhibitor IIa (P
Cells of mouse BALB/C immunized with PI-IIa) and mouse myoma cells P3U1 were fused using polyethylene glycol 4000, and the fusion product was fused with PP I-IIa).
-I-11A strain and I-6D strain were obtained by screening with hybridomas producing specific antibodies against -IIa and cloning by limiting dilution method.
Monoclonal antibody produced by 11A strain 1gM
and monoclonal antibody 1gG_1 produced by the I-6D strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23508589A JPH0398596A (en) | 1989-09-11 | 1989-09-11 | Monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23508589A JPH0398596A (en) | 1989-09-11 | 1989-09-11 | Monoclonal antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0398596A true JPH0398596A (en) | 1991-04-24 |
Family
ID=16980847
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP23508589A Pending JPH0398596A (en) | 1989-09-11 | 1989-09-11 | Monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0398596A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002515454A (en) * | 1998-05-20 | 2002-05-28 | エラスムス ユニフェルシテイト ロッテルダム | Method and means for preventing or treating inflammation or pruritus |
-
1989
- 1989-09-11 JP JP23508589A patent/JPH0398596A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002515454A (en) * | 1998-05-20 | 2002-05-28 | エラスムス ユニフェルシテイト ロッテルダム | Method and means for preventing or treating inflammation or pruritus |
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