JP4016304B2 - Monoclonal antibody, hybrid cell, and method for producing monoclonal antibody - Google Patents

Description

（Ａｃはアセチル基を示す。）
と反応性を有することを特徴とするモノクローナル抗体である。
【００１６】
本発明はＭＧで修飾した蛋白質を調製し、これを抗原とし、該抗原で免疫された恒温動物（ヒトを除く）から得られる抗体産生細胞とミエローマ細胞とを融合させたハイブリッド細胞が産生する、メチルグリオキサール修飾アルギニン誘導体を特異的に認識するモノクローナル抗体であることを特徴とする。
【００１８】
Ｎδ−（５−ヒドロキシ−４，６−ジメチルピリミジン−２−イル）オルニチン（略語：ＡＰ）は、次式（１）で示される化合物である。
【００１９】
【化４】

（Ａｃはアセチル基を示す。）
【００２０】
Ｎδ−（５−ヒドロキシ−５−メチル−４−イミダゾロン−２−イル）オルニチン（略語：５ＨＭＩ）は、次式（２）で示される化合物である。
【００２１】
【化５】

（Ａｃはアセチル基を示す。）
【００２２】
前記式（３）で示される化合物をＭＡ１と称す。
【００２４】
また、本発明は、（１）メチルグリオキサールで修飾した蛋白質を調製し、（２）前記工程で得られた蛋白質を抗原として免疫した恒温動物（ヒトを除く）から得られる抗体産生細胞とミエローマ細胞とを融合させてハイブリッド細胞とし、（３）該ハイブリッド細胞を培養して培養物中からメチルグリオキサール修飾アルギニン誘導体を特異的に認識し、且つ、Ｎδ−（５−ヒドロキシ−４，６−ジメチルピリミジン−２−イル）オルニチン、Ｎδ−（５−ハイドロ−５−メチル−４−イミダゾロン−２−イル）オルニチン、及びＭＡ１と反応性を有するモノクローナル抗体を取得することを特徴とするモノクローナル抗体の製造方法である。
【００２５】
なお、本発明の前記モノクローナル抗体及びその製造方法においては、ＭＧと供にインキュベートされるべき蛋白質はカギアナカサガイのヘモシアニン（略語：ＫＬＨ）であることが望ましい。
【００２６】
【発明の実施の形態】
本発明のモノクローナル抗体は、ＭＧと供にインキュベートした蛋白質を調製し、これを抗原として免疫された恒温動物（ヒトを除く）から得られる抗体産生細胞と、得られた抗体産生細胞を継代培養可能なミエローマ細胞とのハイブリッド細胞を創出し、このハイブリッド細胞の中から目的のモノクローナル抗体のみを産生するハイブリッド細胞をスクリーニングし、このハイブリッド細胞が抗体を産生する環境下で大量培養することにより抗体は製造される。
【００２７】
本発明のモノクローナル抗体の製造に用いることができる抗原は、蛋白質とＭＧの反応物であれば良い。蛋白質としては特に限定されず、例えば、ウシ血清アルブミン（以下、「ＢＳＡ」と略すこともある。）、卵白アルブミン、カギアナカサガイのヘモシアニン（略語：ＫＬＨ）などが挙げられ、ＫＬＨが好ましく使用できる。
【００２８】
ＭＧと蛋白質の反応を行うための具体的な方法としては、例えば、前記蛋白質とＭＧを緩衝液中で混合（攪拌）して得ることが可能である。用いる緩衝液としては、リン酸塩、炭酸塩などの緩衝液が挙げられ、好ましくはリン酸緩衝液が用いられる。緩衝液のｐＨは該反応が進行するｐＨであれば特に限定されないが、ｐＨ６〜１１、好ましくはｐＨ７〜９である。緩衝液の濃度は、該反応が進行する濃度であれば特に限定されないが、１ｍＭ〜０．５Ｍ、好ましくは１０〜１００ｍＭの緩衝液である。この反応過程は、蛋白質中のアミノ酸の減少率で検出することが可能である。アミノ酸の減少率は、例えば、反応前後の試料のＭＧと反応していないアミノ酸をアミノ酸自動分析装置で測定することにより求めることができる。アミノ酸減少率としては、使用する蛋白質により異なるが、アルギニン減少率が１０％以上、好ましくは２０％以上減少することが望ましい。
【００２９】
このようなアミノ酸減少率となる反応条件は、例えば、２５〜４５℃で１時間〜１カ月が好ましく、さらに好ましくは３０〜４０℃で１日〜７日である。
【００３０】
本発明で免疫される動物は恒温動物（ヒトを除く）が使用できる。該免疫動物としては、例えばマウス、ハムスター、ラット、モルモット、ウサギ、イヌ等であれば特に制限されないが、抗体産生細胞を融合するミエローマ細胞がマウス由来のものであるため、好ましくはマウスが使用される。
【００３１】
免疫する方法は、通常の公知の免疫方法を用いることができる。例えば、７ないし３０日、特に１２ないし１６日間隔で２または３回の投与が好ましい。１回の投与量は、免疫される動物により異なるが、例えば、約０．０５〜２ｍｇ程度を目安とする。投与経路は皮下注射、皮内注射、腹膜腔内注射、静脈内注射、筋肉内注射等を選択することができるが、好ましくは腹膜腔、皮下もしくは筋肉内に注射して行う投与形態である。さらに好ましくは、前記投与経路を２ないし３組み合わせた投与経路、例えば、腹膜腔注射、皮下注射および筋肉内注射全ての投与経路を組み合わせるのが好ましい。
【００３２】
なお、この場合、抗原は適当な緩衝液、例えばフロイントの完全アジュバント、フロイントの不完全アジュバント、水酸化アルミニウム等の通常用いられるアジュバントの１種を含有するナトリウムリン酸緩衝液、生理食塩水等に溶解して用いることができるが、上記のようなアジュバントを使用しなくとも良い。ここで、アジュバントとは抗原と共に投与したとき、非特異的にその抗原に対する免疫反応を増強する物質を意味する。
【００３３】
そして、上記の抗原を免疫した恒温動物を７〜３０日間処置せずに放置した後、該恒温動物の血清を少量採取し、抗体価をウエスタンブロット法、凝集法、酵素免疫測定法、一元放射状免疫拡散法等により測定することができる。より簡便には酵素免疫測定法により測定することができる。抗体価が上昇してきたら、状況に応じて抗原の追加投与を適当回数行うことができる。例えば、０．０１〜１ｍｇ、特に、０．０５〜０．５ｍｇの投与量で１もしくは２回の追加投与が行われる。最後の投与の１ないし３０日後、特に好ましくは１〜７日後に免疫した恒温動物から抗体を産生するリンパ球を含む組織を摘出する。摘出する組織は、抗体を産生するリンパ球を含む抹消リンパ系組織ならどこでも良いが、好ましくは脾臓である。
【００３４】
得られた組織は、例えば、「単クローン抗体実験操作入門」（講談社サイエンティフィック 安藤民衛ら １９９１）等に記載されている方法により、継代培養可能な細胞とするために、例えば、仙台ウイルスやポリエチレングリコール存在下、ある種のガン細胞と細胞融合させて、ハイブリッド細胞を得ることができる。ここで用いられるガン細胞は、同じ恒温動物でも同種の恒温動物のガン細胞を用いることが望ましく、例えばマウスを免疫動物として得られた脾臓細胞と融合させる場合、マウスミエローマ細胞を用いることが好ましい。実際に用いられる細胞融合の方法としては、公知の技術（Ｊ． Ｉｍｍｕｎｏｌ． Ｍｅｔｈｏｄ ３９：２８５−３０８，１９８０）を用いることができる。
【００３５】
例えば、免疫されたマウスから得られた脾臓とマウスミエローマ細胞をポリエチレングリコール存在下で融合を行い、ハイブリッド細胞のみが生育可能であるＨＡＴ培地（ヒポキサンチン、アミノプテリン、チミジン添加培地）により選択的にハイブリッド細胞を増殖させ、ハイブリッド細胞がコロニーを形成させた後、培養上清中の抗体をスクリーニングすることで目的の抗体を産生するハイブリッド細胞を得ることができる。
【００３６】
スクリーニングする方法としては、例えば、ウエスタンブロット法あるいは酵素免疫化学的測定法等が挙げられる。また、目的の抗体を産生するハイブリッド細胞は、限界希釈法を繰り返すことにより最終的に単一のハイブリッド細胞を得ることができる。
【００３７】
さらには、これら目的の抗体を産生するハイブリッド細胞は抗体を産生する環境下で大量培養することにより抗体を製造することができる。
【００３８】
さらに、これらの目的とする抗体を産生するハイブリッド細胞が産生した抗体は、例えば遠心分離、硫酸アンモニウムまたはポリエチレングリコールを用いる蛋白質分画、水性二層分配法、ゲル濾過クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー、電気泳動等の蛋白質の一般的な生化学的分離方法を、単独もしくはいくつかの方法を組み合わせて使用することにより精製することができる。
【００３９】
【実施例】
以下、本発明を実施例により具体的に説明する。なお、本発明は本実施例に限定されるものではない。
【００４０】
〔実施例１〕
１．抗原の調製
１００ｍＭのメチルグリオキサール（略語：ＭＧ）に１ｍｇのカギアナカサガイのヘモシアニン（略語：ＫＬＨ）を５０ｍＭリン酸緩衝液（ｐＨ７．２）で溶解した溶液（濃度：１ｍｇ／ｍｌ）を即座に加え、３７℃で３日間反応させてＭＧとＫＬＨのコンジュゲート（略語：ＭＧ−ＫＬＨ）を得て、これを抗原とした。自動アミノ酸分析装置〔ＪＥＯＬ ＪＬＣ−５００、日本分光（株）製〕でＭＧ−ＫＬＨ中の減少したアミノ酸を調べたところ、リジン（以下、「Ｌｙｓ」と略すこともある。）およびアルギニン（以下、「Ａｒｇ」と略すこともある。）は、各々５７％、６５％減少していた。
【００４１】
２．免疫方法
ＭＧ−ＫＬＨ（濃度：１ｍｇ／ｍｌ）は等量のフロイントの完全アジュバントとよく混合してエマルジョンとし、これをマウス（ＢＡＬＢ／ｃ、オス、６〜８週齢）の腹腔内に１００μｌ免疫した。初回免疫から１０〜１４日後、抗原とフロイントの不完全アジュバントをよく混合してエマルジョンとして、追加免疫を行った。追加免疫から３週間後、抗原とリン酸緩衝生理食塩水（以下、「ＰＢＳ」と略す。）を混合して最終免疫を行った。なお、抗体価は、追加免疫の１週間後、マウス眼孔静脈から血液を採取し、得られた血清を用いて酵素免疫化学的方法で抗原に対する抗体が産生していることを確認した。酵素免疫化学的方法では、免疫したマウスから得られた血清と比較対照となる免疫前のマウスから得られた血清の間に比較的大きな抗体価の差異が見られ、免疫後に抗体価の上昇が確認された。
【００４２】
３．抗体価の確認方法
抗体価はＭＧとウシ血清アルブミンを固相とした酵素免疫測定法により確認した。すなわち、抗原の調製に記載した方法と同様にウシ血清アルブミン（以下、「ＢＳＡ」と略すこともある。）とＭＧを反応させて、ＭＧとＢＳＡのコンジュゲート（以下、「ＭＧ−ＢＳＡ」と略すこともある。）を得た。これを９６穴イムノプレートに物理吸着させ、０．０５％Ｔｗｅｅｎ２０−トリス緩衝液（ｐＨ７．４）（以下、「ＴＴＢＳ」と略すこともある。）で３回洗浄した後、１％ＢＳＡを含むトリス緩衝液（ｐＨ７．４）（以下、「ブロッキング液」と略すこともある。）でブロッキングを行った。このプレートのウエルにマウスから得られた血清（１００μｌ／ウエル）を入れて、３７℃で１時間反応させた。ウエルを洗浄後、西洋ワサビペルオキシダーゼで標識されているマウス抗体に対するウサギ抗体をＴＴＢＳで５０００倍希釈した液（１００μｌ／ウエル）（以下、「酵素標識抗体溶液」と略す。）を入れて、３７℃で１時間反応させた。ウエルを洗浄後、Ｏ−フェニレンジアミン（０．４ｍｇ／ｍｌ）および過酸化水素（０．００３％）を含む０．１Ｍクエン酸−リン酸緩衝液（ｐＨ５）（１００μｌ／ウエル）（以下、発色液と略す。）を入れて室温で１５−２０分間反応させた。発色反応は１Ｍの硫酸（５０μｌ／ウエル）（以下、反応停止液と略す。）を入れることで停止し、マイクロプレートリーダーで４９２ｎｍの吸光度を測定して抗体価の確認を行った。なお、ブランクとしては、ＭＧ−ＢＳＡのかわりにＢＳＡを用いたプレートを準備して用いた。
【００４３】
４．細胞融合
免疫したマウスからマウス脾臓を摘出し、よくほぐして脾細胞を得た。得られた脾細胞はＲＰＭＩ−１６４０培地で洗浄した。この洗浄した脾細胞と同様にＲＰＭＩ−１６４０培地でよく洗浄したミエローマ細胞であるマウス６５３細胞（Ｐ３Ｘ６３−Ａｇ８，６５３：ＣＲＬ・１５８０）を細胞数が７：１の割合になるように混和し、培地に対して５０ｗ／ｖ％のポリエチレングリコール１５４０溶液を徐々に加えて５分間混和した。これにＲＰＭＩ−１６４０培地を加えて反応を停止させた後、５分間遠心分離して上清を廃棄した。これにＲＰＭＩ−１６４０培地を加えた後、５分間遠心分離を行い上清を廃棄した。この操作を２回繰り返して細胞を洗浄した。
【００４４】
５．クローニング
前記工程で得られた洗浄細胞に３０ｍｌのＨＡＴ培地を加えて細胞を懸濁し、９６穴マイクロプレートの各ウエルに１００μｌずつ分注し、ＨＡＴ培地により選択的にハイブリッド細胞を増殖させた。融合から１０日後、ＨＴ培地（ＨＡＴ培地からアミノプテリンを除いたもの）でウエル中の１／２量の培養上清を置換した。この操作を２〜３回繰り返した。培養上清については酵素免疫化学的方法により抗体価を確認し、スクリーニングを行った。なお、抗体価の確認方法は、前述の抗体価の確認方法と同様であるが、試験に用いる試料として血清の代わりに得られた培養上清を用いた。スクリーニングにより抗体活性の確認されたウエルの細胞は限界希釈を行い培養し、最終的にはスクリーニングと限界希釈を繰り返すことによりＭＧ修飾蛋白質に対して高い抗体活性を有し、且つ単一の細胞からなるクローン株を得た。該クローン株は、通商産業省工業技術院生命工学工業技術研究所に平成１０年２月２５日に寄託番号（ＦＥＲＭ Ｐ−１６６６５）として寄託されている。
【００４５】
得られたクローン株の生産するモノクローナル抗体は、前記クローン株をＢＡＬＢ／Ｃマウスの腹腔内で増殖させ、その腹水中からプロテイン−ＡセファロースＦＦカラム（ファルマシア社製）を用いてそれぞれを精製した。
【００４６】
〔実施例２〕
前記実施例１のクローン株から得られたＭＧ修飾蛋白質を認識するモノクローナル抗体の内、６Ｂ株のモノクローナル抗体の反応特異性について確認を行った。なお、検討に用いるモノクローナル抗体は、前記の精製したものを用いた。
【００４７】
１．モノクローナル抗体の反応特異性の評価法−１
本発明のモノクローナル抗体の反応特異性を酵素免疫測定法にて評価した。すなわち、９６穴イムノプレートにウエル当たり１００μｌの糖或いは、アルデヒド等と供にインキュベートしたＢＳＡ（４μｇ／ｍｌ）または、銅酸化した低比重リポ蛋白質（４μｇ／ｍｌ）を加え、４℃で一昼夜静置してプレートに物理吸着させ、ウエル当たり３００μｌのＴＴＢＳで３回洗浄した後、１％ＢＳＡ含有ＴＴＢＳもしくは蒸留水で４倍希釈したブロックエースをウエル当たり３００μｌ加えてブロッキングした。このプレートを上記と同様にＴＴＢＳで３回洗浄した後、ウエル当たり１００μｌのＴＴＢＳで希釈した本モノクローナル抗体溶液（１μｇ／ｍｌ）を加えて、３７℃で３時間インキュベートした。このプレートをＴＴＢＳで３回洗浄した後、西洋ワサビペルオキシダーゼで標識されているマウス抗体に対するウサギ抗体をＴＴＢＳで５０００倍希釈した液を入れて３７℃で１時間反応させた。このプレートをＴＴＢＳで３回洗浄した後、ウエル当たり１００μｌの発色液を加えて室温で１５〜２０分間インキュベートした。さらにウエル当たり５０μｌの反応停止液を加えた後、マイクロプレートリーダーで４９２ｎｍの吸光度を測定し、本発明のモノクローナル抗体の反応特異性を評価した。
【００４８】
２．モノクローナル抗体の反応特異性評価法−２
本発明のモノクローナル抗体の反応特異性を酵素免疫測定法にて評価した。すなわち、９６穴イムノプレートにＭＧと供にインキュベートしたＢＳＡを加えてプレートに物理吸着させたプレートのウエルに、本発明によるモノクローナル抗体（１μg／ｍｌ）に終濃度１ｍＭとなるように各種のＭＧ処理アミノ酸（ＭＧ−アミノ酸）を加えたものを試料とする以外は前項に記載の方法と同様にして吸光度を測定し、本発明のモノクローナル抗体の反応特異性を評価した。
【００４９】
３．ＭＧ処理アミノ酸の調製方法
ＭＧ−アミノ酸は、前記実施例１の「抗原の調製」に記載した方法に準じて調製を行った。すなわち、アミノ酸として、Ｎ−α−アセチル−Ｌ−リジン（略語：Ｎａｃｅｔｙｌ−Ｌｙｓ）または、Ｎ−α−アセチル−Ｌ−アルギニン（略語：Ｎａｃｅｔｙｌ−Ａｒｇ）をＭＧと供にインキュベートしてＭＧ−アミノ酸誘導体を調製した。調製したＭＧ−アミノ酸誘導体は、それぞれ、溶媒系として１０％メタノール含有５０ｍＭ酢酸を流速１．０ｍｌ／ｍｉｎとするＤｅｖｅｌｏｓｉｌ ＯＤＳ−ＨＧ−５（８×２５０ｍｍ；Ｎｏｍｕｒａ Ｃｈｅｍｉｃａｌｓ）に供した。溶出プロフィルは、２１５ｎｍの吸光度によりモニターし、ピークを分取した。得られたピークをマススペクトロメーター（ＪＯＥＬ ＪＭＳ−ＤＸ ７０５ ｍａｓｓ ｓｐｅｃｔｒｏｍｅｔｅｒ）およびＮＭＲ（Ｂｒｕｋｅｒ ＡＲＸ−４００ ｓｐｅｃｔｒｏｍｅｔｅｒ）で解析した結果、ＭＧ−（Ｎａｃｅｔｙｌ−Ｌｙｓ）誘導体として、１，３−ジ−Ｎα−アセチルリジノ−４−メチルイミダゾール（略語：ＩＬ）およびＮδ−（カルボキシエチル）−Ｎα−アセチルリジン（略語：ＣＥＬ）、ＭＧ−（Ｎａｃｅｔｙｌ−Ａｒｇ）誘導体として、前記式（１）で示されるＮδ−（５−ヒドロキシ−４，６−ジメチルピリミジン−２−イル）オルニチン（略語：ＡＰ）、前記式（２）で示されるＮδ−（５−ハイドロ−５−メチル−４−イミダゾロン−２−イル）オルニチン（略語：５ＨＭＩ）、および前記式（３）で示されるＭＡ１を得た。
【００５０】
なお、ＩＬは次式（４）で示される化合物である。
【００５１】
【化６】

（Ａｃはアセチル基を示す。）
【００５２】
また、ＣＥＬは次式（５）で示される化合物である。
【００５３】
【化７】

（Ａｃはアセチル基を示す。）
【００５４】
４．モノクローナル抗体の反応特異性−１
前記実施例１のモノクローナル抗体に関して蛋白質に対する反応特異性を評価した。すなわち、ＭＧ、キシロース、フルクトース、グルコース、アラビノース、ヘキサナール、ペンタナール、プロペナール、ノネナール、オクテナール、ヘキセナール、クロトナール、アクロレイン、マロンジアルデヒド（ＭＤＡ）、４−ヒドロキシノネナール（ＨＮＥ）、カルボキシメチルリジン（ＣＭＬ）、銅酸化低比重ＬＤＬ蛋白質（Ｃｕ２＋ＬＤＬ）、フルクトース−６−リン酸（Ｆ６Ｐ）、グルコース−６−リン酸（Ｇ６Ｐ）、未処理ＢＳＡ（Ｎａｔｉｖｅ）などの図１に記載される各種物質と供にインキュベートしたＢＳＡを用い、前記のモノクローナル抗体の反応特異性評価法−１により測定した。その結果を縦軸に物質、横軸に吸光度（Ｏ．Ｄ．）をとったグラフとして図１に示す。ＭＧとインキュベートしたＢＳＡは認識されるものの、他の物質でインキュベートしたＢＳＡは全く認識されなかった。
【００５５】
５．モノクローナル抗体の反応特異性−２
前記実施例１のモノクローナル抗体に関してＭＧ−アミノ酸反応生成物に対する反応特異性を評価した。前記、実施例１、「抗原の調製」の項に示したように、ＭＧと蛋白質を反応させた場合、自動アミノ酸分析装置で測定したＭＧ修飾蛋白質中の減少するアミノ酸は、リジン（以下、「Ｌｙｓ」と略すこともある。）およびアルギニン（以下、「Ａｒｇ」と略すこともある。）である。従って、ＭＧ−アミノ酸誘導体としては、アミノ酸としてＬｙｓおよびＡｒｇを用いて前記実施例１のモノクローナル抗体の反応特異性を評価した。すなわち、Ｎａｃｅｔｙｌ−Ｌｙｓ、Ｎａｃｅｔｙｌ−ＡｒｇおよびＮａｃｅｔｙｌ−ＬｙｓまたはＮａｃｅｔｙｌ−ＡｒｇのＭＧ修飾物（略語：ＭＧ＋Ｎａｃｅｔｙｌ−Ｌｙｓ、ＭＧ＋Ｎａｃｅｔｙｌ−Ａｒｇ）を競合物質として前記のモノクローナル抗体の反応特異性評価法−２により測定した。その結果を縦軸に競合物質、横軸に吸光度（Ｏ．Ｄ．）をとったグラフとして図２に示す。その結果、Ｎａｃｅｔｙｌ−ＡｒｇのＭＧ修飾物を競合物質として添加したもののみ吸光度が減少した。従って本抗体はＭＧ＋Ｎａｃｅｔｙｌ−Ａｒｇは認識するものの、他のＭＧ−アミノ酸誘導体は全く認識しないことが明らかとなった。
【００５６】
６．モノクローナル抗体の反応性−３
前記実施例２の「モノクローナル抗体の反応性−２」から得られた結果をさらに詳細に検討するために、実施例１のモノクローナル抗体に関してＭＧ−アミノ酸誘導体に対する反応特異性を評価した。すなわち、ＭＧ−（Ｎａｃｅｔｙｌ−Ｌｙｓ）誘導体として、１，３−ジ−Ｎα−アセチルリジン−４−メチルイミダゾール（略語：ＩＬ）およびＮδ−（カルボキシエチル）−Ｎα−アセチルリジン（略語：ＣＥＬ）、ＭＧ−（Ｎａｃｅｔｙｌ−Ａｒｇ）誘導体として、Ｎδ−（５−ハイドロ−５−メチル−４−イミダゾロン−２−イル）オルニチン（略語：５ＨＭＩ）、Ｎδ−（５−ヒドロキシ−４，６−ジメチルピリミジン−２−イル）オルニチン（略語、ＡＰ）およびＭＡ１を競合物質として前記のモノクローナル抗体の反応特異性評価法−２により測定した。その結果を縦軸に競合物質、横軸に吸光度（Ｏ．Ｄ．）をとったグラフとして図３に示す。その結果、ＭＧとＡｒｇの反応生成物である、ＭＡ１、５ＨＭＩおよびＡＰを競合物質として添加したもののみ吸光度が減少した。従って本抗体はＭＧとＡｒｇの反応生成物、ＭＧ―アミノ酸誘導体のみを認識することが明らかとなった。
【００５７】
【発明の効果】
以上の説明から明らかなように、本発明によればメチルグリオキサールと蛋白質の反応生成物が結合したときに生じるメチルグリオキサール修飾アルギニン誘導体を特異的に認識するモノクローナル抗体を提供することができる。また、上記実施例から明らかなように、本発明のモノクローナル抗体は、認識部位が分子構造レベルで限定されているので、臨床診断、分析等において有用性が有り、糖化蛋白質等の疾患・病態との関連性の解明のための一助となることが期待される。
【図面の簡単な説明】
【図１】種々の物質をＢＳＡと供にインキュベートしたＢＳＡ修飾体に対する本発明のモノクローナル抗体の反応性について、酵素免疫学的測定法により測定した結果を示すグラフである。
【図２】ＭＧ−アミノ酸誘導体に対する本発明のモノクローナル抗体の認識構造について、酵素免疫学的測定法により測定した結果を示すグラフである。
【図３】本発明のモノクローナル抗体の認識構造について、酵素免疫学的測定法により測定した結果を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a monoclonal antibody and a production method thereof, in particular, a monoclonal antibody having high specificity for a reaction product of methylglyoxal and an amino acid, peptide or protein arginine residue, a hybrid cell producing the monoclonal antibody, and a monoclonal antibody It relates to a manufacturing method. More specifically, the present invention relates to a monoclonal antibody that specifically recognizes a methylglyoxal-modified arginine derivative, a hybrid cell that produces the monoclonal antibody, and a method for producing the monoclonal antibody.
[0002]
[Prior art]
The amino group of an amino acid, peptide or protein is condensed non-enzymatically with an aldehyde group of a reducing sugar, and is non-enzymatically saccharified to be glycated amino acid, glycated peptide or glycated protein (hereinafter abbreviated as “glycated protein etc.”). It is known that The saccharification reaction is called the Maillard reaction, and this reaction proceeds in vivo, and is known to be involved in the development of aging and diabetic complications (Bio Industry vol. 13, No. 7, p14). , 1996).
[0003]
Maillard reaction can be divided into early and late stage reactions. In the first-stage reaction, the side chain amino group or N-terminal amino group of the protein reacts with the carbonyl group of the sugar to form a Schiff base, and then the Amadori rearrangement compound is formed. As the early stage reaction product existing in the living body, for example, hemoglobin A1C, glycated albumin and the like are known, and are known to be involved in various pathological conditions, particularly diabetes.
[0004]
In the latter stage reaction, after the above-mentioned first stage reaction, further through complicated reactions such as oxidation, dehydration, condensation, and cyclization, (1) fluorescence, (2) browning, and (3) intramolecular / molecular A late reaction product (advanced glycation end products. Hereinafter abbreviated as “AGE”) having at least one of the characteristics of intercrosslinking and (4) biological recognition is generated.
[0005]
As the AGE structure, carboxymethyl lysine, pyralin, pentosidine, croslin, or X1 has been proposed.
[0006]
By the way, an antibody that specifically reacts with a glycated protein or the like and whose specific recognition site is specified at the molecular structure level is detected accurately and precisely when the antibody is used for detection of an antigenic substance. Therefore, it is useful in clinical diagnosis and analysis. As a method for detecting an antigenic substance, any immunochemical detection method can be used, and specific examples include an immunohistological method and an enzyme immunoassay method. Accordingly, there is a strong demand for the appearance of antibodies that specifically recognize such specific molecular structures.
[0007]
From such a background, an antibody against the AGE structure is prepared, and according to immunological studies, it is known to be positive for aging / diabetes, diabetic nephropathy or mammalian lens crystallin. (J. Clin. Invest., 85.380-384, 1990, J. Biol. Chem., 263, 3758-3764, 1989, J. Clin. Invest., 89.1102-1111, 1992). Japanese Patent Application Laid-Open No. 9-178740 describes the use of an antibody against carboxymethyllysine as a marker for diabetes or diabetic complications.
[0008]
On the other hand, methylglyoxal (hereinafter sometimes abbreviated as “MG”) is produced in vivo, particularly in blood by non-enzymatic or enzymatic catabolism of triose phosphate, dihydroxyacetone phosphate, or glyceraldehyde-3-phosphate. It is known to be present in relatively large amounts, and it has been reported that serum levels in diabetic patients are high, or that they are present in large amounts in the lens of rats that are induced by streptozotocin (Biochem). Pharmacol. 46, 805-811, 1993, Clin. Sci., 87, 21-29, 1994). In addition, MG not only can function as a direct mediator to generate fluorescent products by reacting with proteins at in vivo concentration levels, but also reports its relevance to diabetes and aging. (Biochim. Biophys. Acta., 1270, 36-43, 1995, J. Biol. Chem., 269, 32299-32305, 1994, J. Biol. Chem., 267, 4364-4369, 1992).
[0009]
Therefore, specifically detecting a substance or site having an AGE structure produced by the reaction of MG and protein is considered useful for clinical diagnosis, analysis, and the like.
[0010]
Under such circumstances, immunohistological studies using polyclonal antibodies against amino acids modified with MG have reported that human atherosclerotic lesions have sites that are strongly stained with the polyclonal antibodies ( FEBS Letters, 410, 313-318, 1997).
[0011]
However, the above antibody is a polyclonal antibody. When obtaining an antibody, (1) a large amount of antigen is required, (2) antibody titer varies from production to production, and (3) economical efficiency is low. In addition, the specific recognition site of the antibody is not specified at the molecular structure level, causing problems such as unclear measurement targets. Specific molecular structures that do not have such disadvantages are specifically recognized. There is a need for monoclonal antibodies.
[0012]
By the way, the reaction of MG and protein is already known (J. Biol. Chem., Vol. 269. p32299-32305.1994, J, Prot. Chem., Vol. 14. p359-3721.995), MG. It is known that MG reacts with protein, peptide or amino acid, MG reacts with protein, peptide arginine, or single arginine. Therefore, it is clinically or analytically possible to detect glycated proteins and the like by detecting substances or sites having methylglyoxal-modified arginine derivatives produced by the reaction of MG and protein, arginine in peptides, or single arginine. It is useful above.
[0013]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a monoclonal antibody that specifically reacts with a glycated protein, etc., and whose specific recognition site is specified at the molecular structure level, and is useful in clinical diagnosis, analysis, etc. It is.
[0014]
[Means for Solving the Invention]
As a result of investigation in view of the above problems, the present inventors have incubated hemocyanin of scallop limpet (hereinafter sometimes abbreviated as “KLH”) together with methylglyoxal (abbreviation: MG) and in MG. A monoclonal antibody that specifically recognizes a methylglyoxal-modified arginine derivative is produced by a hybrid cell of antibody-producing cells and myeloma cells obtained from a thermostatized animal (excluding humans) immunized with a modified KLH as an antigen. As a result, the present invention was completed.
[0015]
That is, the present invention specifically recognizes a methylglyoxal-modified arginine derivative, and Nδ- (5-hydroxy-4,6-dimethylpyrimidin-2-yl) ornithine, Nδ- (5-hydro-5-methyl- 4-Imidazolone-2-yl) ornithine, and a compound represented by the following formula (3)

(Ac represents an acetyl group.)
It is a monoclonal antibody characterized by having reactivity.
[0016]
The present invention prepares a protein modified with MG, uses this as an antigen, and produces a hybrid cell obtained by fusing an antibody-producing cell obtained from a homeothermic animal (excluding human) immunized with the antigen and a myeloma cell. A monoclonal antibody that specifically recognizes a methylglyoxal-modified arginine derivative.
[0018]
Nδ- (5-hydroxy-4,6-dimethylpyrimidin-2-yl) ornithine (abbreviation: AP) is a compound represented by the following formula (1).
[0019]
[Formula 4]

(Ac represents an acetyl group.)
[0020]
Nδ- (5-hydroxy-5-methyl-4-imidazolon-2-yl) ornithine (abbreviation: 5HMI) is a compound represented by the following formula (2).
[0021]
[Chemical formula 5]

(Ac represents an acetyl group.)
[0022]
The compound represented by the formula (3) is referred to as MA1.
[0024]
The present invention also provides (1) a protein modified with methylglyoxal, and (2) an antibody-producing cell and a myeloma cell obtained from a homeothermic animal (excluding humans) immunized with the protein obtained in the above step as an antigen. And (3) culturing the hybrid cell to specifically recognize the methylglyoxal-modified arginine derivative from the culture, and Nδ- (5-hydroxy-4,6-dimethylpyrimidine -2-yl) ornithine, Nδ- (5-hydro-5-methyl-4-imidazolon-2-yl) ornithine, and monoclonal antibody reactive with MA1 It is.
[0025]
In the monoclonal antibody and the method for producing the same according to the present invention, the protein to be incubated with MG is preferably limpet hemocyanin (abbreviation: KLH).
[0026]
DETAILED DESCRIPTION OF THE INVENTION
The monoclonal antibody of the present invention is prepared by preparing a protein incubated with MG, and using this as an antigen, antibody-producing cells obtained from a thermostatized animal (excluding humans) and the obtained antibody-producing cells are subcultured. By creating a hybrid cell with a possible myeloma cell, screening a hybrid cell that produces only the target monoclonal antibody from this hybrid cell, and by culturing in large quantities in an environment where this hybrid cell produces the antibody, Manufactured.
[0027]
The antigen that can be used for the production of the monoclonal antibody of the present invention may be a reaction product of protein and MG. The protein is not particularly limited, and examples thereof include bovine serum albumin (hereinafter sometimes abbreviated as “BSA”), ovalbumin, limpet hemocyanin (abbreviation: KLH), and KLH is preferably used. .
[0028]
As a specific method for performing the reaction between MG and protein, for example, the protein and MG can be mixed (stirred) in a buffer solution. Examples of the buffer used include buffers such as phosphates and carbonates, and phosphate buffers are preferably used. The pH of the buffer solution is not particularly limited as long as the reaction proceeds, but it is pH 6 to 11, preferably pH 7 to 9. The concentration of the buffer is not particularly limited as long as the reaction proceeds, but it is 1 mM to 0.5 M, preferably 10 to 100 mM. This reaction process can be detected by the reduction rate of amino acids in the protein. The reduction rate of amino acids can be determined, for example, by measuring amino acids that have not reacted with MG of the sample before and after the reaction using an amino acid automatic analyzer. The amino acid reduction rate varies depending on the protein used, but it is desirable that the arginine reduction rate is reduced by 10% or more, preferably 20% or more.
[0029]
The reaction conditions that give such an amino acid reduction rate are preferably, for example, 25 to 45 ° C. for 1 hour to 1 month, and more preferably 30 to 40 ° C. for 1 day to 7 days.
[0030]
As the animal to be immunized in the present invention, a thermostat animal (excluding humans) can be used. The immunized animal is not particularly limited as long as it is, for example, mouse, hamster, rat, guinea pig, rabbit, dog, etc. However, since myeloma cells that fuse antibody-producing cells are derived from mice, mice are preferably used. The
[0031]
As a method for immunization, an ordinary known immunization method can be used. For example, administration of 2 or 3 times at intervals of 7 to 30 days, particularly 12 to 16 days is preferable. A single dose varies depending on the animal to be immunized, but is, for example, about 0.05 to 2 mg. The administration route can be selected from subcutaneous injection, intradermal injection, intraperitoneal injection, intravenous injection, intramuscular injection, and the like. The administration form is preferably performed by injection into the peritoneal cavity, subcutaneously or intramuscularly. More preferably, it is preferable to combine all the administration routes, such as peritoneal cavity injection, subcutaneous injection and intramuscular injection, by combining 2 to 3 administration routes.
[0032]
In this case, the antigen is contained in an appropriate buffer such as Freund's complete adjuvant, Freund's incomplete adjuvant, sodium phosphate buffer containing one kind of commonly used adjuvants such as aluminum hydroxide, physiological saline and the like. Although it can be used by dissolving, it is not necessary to use the above adjuvant. Here, an adjuvant means a substance that, when administered together with an antigen, nonspecifically enhances an immune response to the antigen.
[0033]
And after leaving the isothermal animal immunized with the above antigen untreated for 7 to 30 days, a small amount of serum of the isothermal animal is collected, and the antibody titer is determined by Western blotting, agglutination method, enzyme immunoassay method, one-dimensional radial It can be measured by an immunodiffusion method or the like. More simply, it can be measured by enzyme immunoassay. If the antibody titer increases, additional administration of antigen can be performed an appropriate number of times depending on the situation. For example, one or two additional administrations are performed at a dose of 0.01 to 1 mg, particularly 0.05 to 0.5 mg. From 1 to 30 days after the last administration, particularly preferably after 1 to 7 days, a tissue containing antibody-producing lymphocytes is removed from the immunized animal. The tissue to be removed may be any peripheral lymphoid tissue containing antibody-producing lymphocytes, but is preferably the spleen.
[0034]
The obtained tissue can be transformed into cells that can be subcultured by, for example, the method described in “Introduction to Monoclonal Antibody Experimental Manipulation” (Kodansha Sangyo Ando et al. 1991). Hybrid cells can be obtained by cell fusion with certain cancer cells in the presence of virus or polyethylene glycol. The cancer cells used here are preferably the same isothermal animal cancer cells of the same species, and for example, when a mouse is fused with a spleen cell obtained as an immunized animal, a mouse myeloma cell is preferably used. As a method of cell fusion actually used, a known technique (J. Immunol. Method 39: 285-308, 1980) can be used.
[0035]
For example, spleen obtained from an immunized mouse and mouse myeloma cells are fused in the presence of polyethylene glycol, and selectively by a HAT medium (medium supplemented with hypoxanthine, aminopterin, and thymidine) in which only hybrid cells can grow. After the hybrid cells are grown and the hybrid cells form colonies, the antibodies in the culture supernatant are screened to obtain hybrid cells that produce the target antibody.
[0036]
Examples of screening methods include Western blotting or enzyme immunochemical measurement. A hybrid cell producing the target antibody can be finally obtained as a single hybrid cell by repeating the limiting dilution method.
[0037]
Furthermore, the hybrid cells producing these desired antibodies can be produced by culturing them in a large amount in an antibody-producing environment.
[0038]
Furthermore, antibodies produced by hybrid cells that produce these desired antibodies include, for example, centrifugation, protein fractionation using ammonium sulfate or polyethylene glycol, aqueous two-layer partitioning, gel filtration chromatography, ion exchange chromatography, affinity The protein can be purified by using general biochemical separation methods such as chromatography and electrophoresis alone or in combination of several methods.
[0039]
【Example】
Hereinafter, the present invention will be specifically described by way of examples. In addition, this invention is not limited to a present Example.
[0040]
[Example 1]
1. Preparation of antigen Immediately a solution (concentration: 1 mg / ml) of 100 mg methylglyoxal (abbreviation: MG) in which 1 mg limpet hemocyanin (abbreviation: KLH) was dissolved in 50 mM phosphate buffer (pH 7.2). In addition, it was reacted at 37 ° C. for 3 days to obtain a conjugate of MG and KLH (abbreviation: MG-KLH), which was used as an antigen. When the reduced amino acid in MG-KLH was examined with an automatic amino acid analyzer [JEOL JLC-500, manufactured by JASCO Corporation], lysine (hereinafter sometimes abbreviated as “L ys ”) and arginine (hereinafter referred to as “L ys ”). , And may be abbreviated as “Arg”.) Were reduced by 57% and 65%, respectively.
[0041]
2. Immunization method MG-KLH (concentration: 1 mg / ml) was mixed well with an equal volume of Freund's complete adjuvant to form an emulsion, which was immunized with 100 μl intraperitoneally into mice (BALB / c, male, 6-8 weeks old). did. Ten to 14 days after the first immunization, the antigen and Freund's incomplete adjuvant were mixed well to give an emulsion as a booster immunization. Three weeks after the booster immunization, the antigen and phosphate buffered saline (hereinafter abbreviated as “PBS”) were mixed for final immunization. In addition, the antibody titer was confirmed that an antibody against the antigen was produced by an enzyme immunochemical method using blood obtained by collecting blood from the murine ocular vein one week after the booster immunization. In the enzyme immunochemical method, a relatively large antibody titer difference was observed between the serum obtained from the immunized mouse and the serum obtained from the pre-immunized mouse as a comparative control, and the antibody titer increased after immunization. confirmed.
[0042]
3. Method for confirming antibody titer The antibody titer was confirmed by enzyme immunoassay using MG and bovine serum albumin as solid phases. That is, bovine serum albumin (hereinafter sometimes abbreviated as “BSA”) and MG are reacted in the same manner as described in the preparation of the antigen, and a conjugate of MG and BSA (hereinafter referred to as “MG-BSA”). It may be omitted.) This was physically adsorbed on a 96-well immunoplate, washed 3 times with 0.05% Tween 20-Tris buffer (pH 7.4) (hereinafter sometimes abbreviated as “TTBS”), and then contains 1% BSA. Blocking was performed with Tris buffer (pH 7.4) (hereinafter, sometimes abbreviated as “blocking solution”). Sera obtained from mice (100 μl / well) was added to the wells of this plate and reacted at 37 ° C. for 1 hour. After washing the wells, a solution (100 μl / well) of a rabbit antibody against mouse antibody labeled with horseradish peroxidase diluted 5000 times with TTBS (hereinafter abbreviated as “enzyme-labeled antibody solution”) was added at 37 ° C. For 1 hour. After washing the well, 0.1 M citrate-phosphate buffer solution (pH 5) (100 μl / well) containing O-phenylenediamine (0.4 mg / ml) and hydrogen peroxide (0.003%) (hereinafter, color development) Abbreviated as liquid) and allowed to react at room temperature for 15-20 minutes. The color reaction was stopped by adding 1 M sulfuric acid (50 μl / well) (hereinafter abbreviated as reaction stop solution), and the antibody titer was confirmed by measuring the absorbance at 492 nm with a microplate reader. In addition, as a blank, the plate which used BSA instead of MG-BSA was prepared and used.
[0043]
4). The mouse spleen was removed from the cell fusion immunized mouse and loosened to obtain spleen cells. The obtained splenocytes were washed with RPMI-1640 medium. Similar to the washed spleen cells, mouse 653 cells (P3X63-Ag8,653: CRL · 1580), which are myeloma cells thoroughly washed with RPMI-1640 medium, were mixed so that the cell number was 7: 1. A 50 w / v% polyethylene glycol 1540 solution was gradually added to the medium and mixed for 5 minutes. RPMI-1640 medium was added thereto to stop the reaction, and then centrifuged for 5 minutes to discard the supernatant. After RPMI-1640 medium was added thereto, the mixture was centrifuged for 5 minutes and the supernatant was discarded. This operation was repeated twice to wash the cells.
[0044]
5). Cloning 30 ml of HAT medium was added to the washed cells obtained in the previous step to suspend the cells, and 100 μl was dispensed into each well of a 96-well microplate, and hybrid cells were selectively grown in the HAT medium. Ten days after the fusion, ½ volume of the culture supernatant in the well was replaced with HT medium (HAT medium minus aminopterin). This operation was repeated 2-3 times. The culture supernatant was screened after confirming the antibody titer by an enzyme immunochemical method. The method for confirming the antibody titer is the same as the method for confirming the antibody titer described above, but the culture supernatant obtained instead of serum was used as a sample used in the test. Cells in wells in which antibody activity has been confirmed by screening are subjected to limiting dilution and cultured. Finally, screening and limiting dilution are repeated to have high antibody activity against MG-modified proteins, and from single cells. A clone strain was obtained. The clone strain was deposited at the Biotechnology Institute of Industrial Technology, Ministry of International Trade and Industry on February 25, 1998 as a deposit number (FERM P-16665).
[0045]
The monoclonal antibodies produced by the obtained clonal strains were grown in the peritoneal cavity of BALB / C mice and purified from the ascites using a protein-A Sepharose FF column (Pharmacia).
[0046]
[Example 2]
Among the monoclonal antibodies recognizing the MG-modified protein obtained from the clone strain of Example 1, the reaction specificity of the 6B strain monoclonal antibody was confirmed. The purified monoclonal antibody was used for the examination.
[0047]
1. Evaluation Method of Reaction Specificity of Monoclonal Antibody-1
The reaction specificity of the monoclonal antibody of the present invention was evaluated by enzyme immunoassay. That is, BSA (4 μg / ml) or copper-oxidized low-density lipoprotein (4 μg / ml) incubated with 100 μl sugar or aldehyde or the like per well was added to a 96-well immunoplate and allowed to stand overnight at 4 ° C. The plate was physically adsorbed on the plate, washed 3 times with 300 μl TTBS per well, and then blocked by adding 300 μl per well of Block Ace diluted with 1% BSA-containing TTBS or distilled water 4 times. The plate was washed 3 times with TTBS in the same manner as described above, and the monoclonal antibody solution (1 μg / ml) diluted with 100 μl of TTBS was added per well, followed by incubation at 37 ° C. for 3 hours. The plate was washed 3 times with TTBS, and then a solution obtained by diluting a rabbit antibody against a mouse antibody labeled with horseradish peroxidase 5000 times with TTBS was added and reacted at 37 ° C. for 1 hour. The plate was washed 3 times with TTBS, and then 100 μl of color developing solution was added per well and incubated at room temperature for 15-20 minutes. Further, 50 μl of reaction stop solution was added per well, and then the absorbance at 492 nm was measured with a microplate reader to evaluate the reaction specificity of the monoclonal antibody of the present invention.
[0048]
2. Method for evaluating reaction specificity of monoclonal antibody-2
The reaction specificity of the monoclonal antibody of the present invention was evaluated by enzyme immunoassay. That is, various MG treatments were carried out so that the final concentration of the monoclonal antibody according to the present invention (1 μg / ml) was 1 mM in the well of the plate which was physically adsorbed on the plate by adding BSA incubated with MG to 96-well immunoplate. Absorbance was measured in the same manner as described in the previous section except that an amino acid (MG-amino acid) added was used as a sample, and the reaction specificity of the monoclonal antibody of the present invention was evaluated.
[0049]
3. Preparation of MG-treated amino acid MG-amino acid was prepared according to the method described in “Preparation of antigen” in Example 1 above. That is, N-α-acetyl-L-lysine (abbreviation: Nacetyl-Lys) or N-α-acetyl-L-arginine (abbreviation: Nacetyl-Arg) is incubated with MG as an MG-amino acid. Derivatives were prepared. Each of the prepared MG-amino acid derivatives was subjected to Develosil ODS-HG-5 (8 × 250 mm; Nomura Chemicals) using 50 mM acetic acid containing 10% methanol as a solvent system at a flow rate of 1.0 ml / min. The elution profile was monitored by absorbance at 215 nm, and peaks were collected. The obtained peak was analyzed by mass spectrometer (JOEL JMS-DX 705 mass spectrometer) and NMR (Bruker ARX-400 spectrometer). As a result, 1,3-di-Nα-acetylridino was obtained as an MG- (Nacetyl-Lys) derivative. As 4-methylimidazole (abbreviation: IL) and Nδ- (carboxyethyl) -Nα-acetyllysine (abbreviation: CEL), MG- (Nacetyl-Arg) derivatives, Nδ- (5 -Hydroxy-4,6-dimethylpyrimidin-2-yl) ornithine (abbreviation: AP), Nδ- (5-hydro-5-methyl-4-imidazolon-2-yl) ornithine represented by the formula (2) ( Abbreviation: 5HMI) and the above formula (3) MA1 was obtained.
[0050]
IL is a compound represented by the following formula (4).
[0051]
[Chemical 6]

(Ac represents an acetyl group.)
[0052]
CEL is a compound represented by the following formula (5).
[0053]
[Chemical 7]

(Ac represents an acetyl group.)
[0054]
4). Reaction specificity of monoclonal antibody-1
The specificity of the monoclonal antibody of Example 1 for the protein was evaluated. That is, MG, xylose, fructose, glucose, arabinose, hexanal, pentanal, propenal, nonenal, octenal, hexenal, crotonal, acrolein, malondialdehyde (MDA), 4-hydroxynonenal (HNE), carboxymethyllysine (CML), Incubation with various substances shown in FIG. 1, such as copper oxide low density LDL protein (Cu2 + LDL), fructose-6-phosphate (F6P), glucose-6-phosphate (G6P), untreated BSA (Native) The BSA was measured by the above-described monoclonal antibody reaction specificity evaluation method-1. The results are shown in FIG. 1 as a graph with the substance on the vertical axis and the absorbance (OD) on the horizontal axis. Although BSA incubated with MG was recognized, BSA incubated with other substances was not recognized at all.
[0055]
5). Reaction specificity of monoclonal antibody-2
With respect to the monoclonal antibody of Example 1, the reaction specificity for the MG-amino acid reaction product was evaluated. As shown in Example 1, “Preparation of antigen”, when MG and protein are reacted, the amino acid decreased in the MG-modified protein measured by an automatic amino acid analyzer is lysine (hereinafter, “ L ys ") and arginine (hereinafter sometimes abbreviated as" Arg "). Therefore, as the MG-amino acid derivative, the reaction specificity of the monoclonal antibody of Example 1 was evaluated using Lys and Arg as amino acids. That is, Nacetyl-Lys, Nacetyl-Arg and Nacetyl-Lys or MG modified product of Nacetyl-Arg (abbreviation: MG + Nacetyl-Lys, MG + Nacetyl-Arg) measured by the above-mentioned monoclonal antibody reaction specificity evaluation method-2 did. The results are shown in FIG. 2 as a graph in which the vertical axis represents the competitor and the horizontal axis represents the absorbance (OD). As a result, the absorbance was decreased only for those in which Nacethyl-Arg-modified MG was added as a competitor. Therefore, it was revealed that this antibody recognizes MG + Nacethyl-Arg but does not recognize other MG-amino acid derivatives at all.
[0056]
6). Monoclonal antibody reactivity-3
In order to examine the results obtained from “Reactivity of monoclonal antibody-2” in Example 2 in more detail, the specificity of the monoclonal antibody of Example 1 for the MG-amino acid derivative was evaluated. That is, as MG- (Nacetyl-Lys) derivatives, 1,3-di-Nα-acetyllysine-4-methylimidazole (abbreviation: IL) and Nδ- (carboxyethyl) -Nα-acetyllysine (abbreviation: CEL), As MG- (Nacetyl-Arg) derivatives, Nδ- (5-hydro-5-methyl-4-imidazolon-2-yl) ornithine (abbreviation: 5HMI), Nδ- (5-hydroxy-4,6-dimethylpyrimidine- 2-Ill) ornithine (abbreviation, AP) and MA1 were used as competitors and measured by the above-described monoclonal antibody reaction specificity evaluation method-2. The results are shown in FIG. 3 as a graph in which the vertical axis represents the competitor and the horizontal axis represents the absorbance (OD). As a result, the absorbance decreased only when MA1, 5HMI and AP, which are reaction products of MG and Arg, were added as competitors. Therefore, it was revealed that this antibody recognizes only the reaction product of MG and Arg and the MG-amino acid derivative.
[0057]
【The invention's effect】
As is clear from the above description, according to the present invention, a monoclonal antibody that specifically recognizes a methylglyoxal-modified arginine derivative produced when a reaction product of methylglyoxal and a protein binds can be provided. Further, as is clear from the above examples, the monoclonal antibody of the present invention has usefulness in clinical diagnosis, analysis and the like because the recognition site is limited at the molecular structure level, and it is useful for diseases and pathologies such as glycated proteins. This is expected to help elucidate the relationship between
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the results of measuring the reactivity of a monoclonal antibody of the present invention against a modified BSA obtained by incubating various substances with BSA by an enzyme immunoassay.
FIG. 2 is a graph showing the results of measuring the recognition structure of the monoclonal antibody of the present invention against MG-amino acid derivative by an enzyme immunoassay.
FIG. 3 is a graph showing the results of measuring the recognition structure of the monoclonal antibody of the present invention by enzyme immunoassay.

Claims (6)

Specific recognition of methylglyoxal-modified arginine derivatives, and Nδ- (5-hydroxy-4,6-dimethylpyrimidin-2-yl) ornithine, Nδ- (5-hydro-5-methyl-4-imidazolone-2 -Il) ornithine and a compound represented by the following formula (3)

(Ac represents an acetyl group.)
Monoclonal antibody characterized by having reactivity with.   The monoclonal antibody is obtained from a hybrid cell in which a protein modified with methylglyoxal is used as an antigen, and an antibody-producing cell obtained from a constant temperature animal (excluding humans) immunized with the antigen and myeloma cell is fused. The monoclonal antibody according to claim 1.   The monoclonal antibody according to claim 2, wherein the antigen is a limpet hemocyanin modified with methylglyoxal.   A hybrid cell capable of producing the monoclonal antibody according to claim 1. (1) Prepare a protein modified with methylglyoxal,
(2) A hybrid cell obtained by fusing an antibody-producing cell obtained from a constant temperature animal (excluding human) immunized with the protein obtained in the above step as an antigen and a myeloma cell,
(3) The hybrid cell is cultured to specifically recognize the methylglyoxal-modified arginine derivative from the culture, and Nδ- (5-hydroxy-4,6-dimethylpyrimidin-2-yl) ornithine, Nδ- (5-Hydroxy-5-methyl-4-imidazolon-2-yl) ornithine and a compound represented by the following formula (3)

(Ac represents an acetyl group.)
A method for producing a monoclonal antibody, comprising obtaining a monoclonal antibody having reactivity with.   6. The method for producing a monoclonal antibody according to claim 5, wherein the antigen is a limpet hemocyanin modified with methylglyoxal.

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