JP4823465B2 - Antibody specifically binding to human HMG-1 and method and reagent for immunoassay of human HMG-1 using this antibody - Google Patents

Description

【０１８８】
４．まとめ
前記の表より、各々のウシＨＭＧ−１濃度において、生理食塩水で希釈した血清を含まない試料の測定値（吸光度）と、ヒト血清で希釈した血清試料の測定値（吸光度）は、ほとんど同じであることが分かる。
【０１８９】
血清は種々の成分を含み複雑な組成のものであるが、本発明の免疫学的測定試薬及び免疫学的測定方法は、このような血清又は血清を含む試料においても、ＨＭＧ−１の濃度を正確に測定することができるものであることが確かめられた。
【０１９０】
なお、先に述べたように、前記測定を行った本発明の免疫学的測定試薬及び免疫学的測定方法に使用した抗体はいずれも、ヒトとウシとで全くアミノ酸配列が同一な箇所を免疫原に使用して作成したものであり、そして、実施例７において本発明の免疫学的測定試薬及び免疫学的測定方法は、前記ヒトＨＭＧ−１も前記ウシＨＭＧ−１も正確に測定できることが確かめられているので、例え、試料としてウシＨＭＧ−１の代わりにヒトＨＭＧ−１を血清で希釈したものを用いたとしても、同じ結果が得られることは明白である。
【０１９１】
〔実施例９〕（抗体のヒトＨＭＧ−１及びヒトＨＭＧ−２との反応性の確認）
実施例４で調製したヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）、実施例４で調製したヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（モノクローナル抗体）、及び参考例４で調製したヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（ポリクローナル抗体）のそれぞれについて、ヒトＨＭＧ−１及びヒトＨＭＧ−２の各々との反応性をウエスタンブロット法により確かめた。
【０１９２】
１．ウエスタンブロット法
（１）ヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（実施例４で調製したモノクローナル抗体）
▲１▼ ヒト血清を、タイタン・ジェル・リポタンパク質電気泳動キット（ヘレナ研究所社製）を用いて電気泳動を行った。なお、支持体はアガロースゲルであり、これに前記のヒト血清の２μＬを接触させた。
▲２▼ そして、泳動緩衝液としてバルビタール緩衝液（ｐＨ８．８）を使用して、電圧９０Ｖで７５分間通電して電気泳動を行った。なお、この電気泳動は、同じものを２セット用意して行い、以下の操作も同様に行った。
▲３▼ 前記▲２▼の電気泳動の後の転写は、ノバ・ブロット・エレクトロフォレティック・トランスファー・キット（ファルマシア−エルケービー社製）を用いて、その使用説明書に従い、ドライ方式で行った。
【０１９３】
まず、前記▲２▼において電気泳動を行ったアガロースゲルを転写用装置上に置いた。次に、このアガロースゲルの上に、９ｃｍ×９ｃｍのニトロセルロース膜（バイオラッド社製）を重ね、４８ｍＭトリス（ヒドロキシメチル）アミノメタン、３９ｍＭグリシン、０．０３７５％（Ｗ／Ｖ）ドデシル硫酸ナトリウム（ＳＤＳ）及び２０％（Ｖ／Ｖ）メタノールよりなる転写用緩衝液を用いて、電流６５ｍＡで２時間転写を行った。
▲４▼ この転写を行ったニトロセルロース膜を、１％ＢＳＡを含むリン酸緩衝生理食塩水（５．５９ｍＭリン酸水素二ナトリウム、１．４７ｍＭリン酸二水素カリウム、１３７ｍＭ塩化ナトリウム及び２．６８ｍＭ塩化カリウムを含む水溶液（ｐＨ７．２））の２０ｍＬに４℃で１晩浸漬して、ブロッキングを行った。
▲５▼ 次に、これを洗浄液（０．０５％ツイーン２０（Ｔｗｅｅｎ２０）を含むリン酸緩衝生理食塩水）の２０ｍＬ中で１０分間振とう洗浄を行った。この操作を３回行った。
▲６▼ 実施例４で調製した、ヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）を２０ｍＬのリン酸緩衝生理食塩水に８０μｇ溶解し、この溶液に前記▲５▼の操作を行ったニトロセルロース膜を室温で２時間浸漬して反応させた。
▲７▼ 前記▲６▼の操作を行ったニトロセルロース膜を、２０ｍＬの洗浄液中で１０分間振とう洗浄を行った。これを３回行った。
▲８▼ 次に、パーオキシダーゼ標識抗マウスＩｇＧ抗体（ダコ社製）を、３％ＢＳＡを含むリン酸緩衝生理食塩水で５００倍希釈をして、２０ｍＬの溶液を調製し、これに前記▲７▼のニトロセルロース膜を室温で２時間浸漬して反応させた。
▲９▼ このニトロセルロース膜を、２０ｍＬの洗浄液中で１０分間振とう洗浄を行った。この操作を３回行った。
[10] ０．０２５％の３，３’−ジアミノベンジジン四塩酸塩及び０．０１％過酸化水素を含むリン酸緩衝生理食塩水の２０ｍＬに、前記▲９▼のニトロセルロース膜を室温で１５分間浸漬して発色させた。
【０１９４】
以上の操作により、実施例４で調製した、ヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）におけるウエスタンブロット法の結果を得た。
【０１９５】
（２）ヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（実施例４で調製したモノクローナル抗体）
前記（１）の▲６▼における「実施例４で調製した、ヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）」を、「実施例４で調製した、ヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（モノクローナル抗体）」に変えること以外は、前記（１）の▲１▼〜[10]の通りに操作を行い、実施例４で調製した、ヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（モノクローナル抗体）におけるウエスタンブロット法の結果を得た。
【０１９６】
（３）ヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（参考例４で調製したポリクローナル抗体）
前記（１）の▲６▼における「実施例４で調製した、ヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）」を、「参考例４で調製した、ヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（ポリクローナル抗体）」に変えること以外は、前記（１）の▲１▼〜[10]の通りに操作を行い、参考例４で調製した、ヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（ポリクローナル抗体）におけるウエスタンブロット法の結果を得た。
【０１９７】
２．実験結果
（１）ウエスタンブロット法の結果
前記１の（１）、（２）及び（３）におけるウエスタンブロット法の結果を図４に示した。
なお、この図において、「ＭＡ１」はヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（実施例４で調製したモノクローナル抗体）における結果であり、「ＭＡ２」はヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（実施例４で調製したモノクローナル抗体）における結果であり、「ＰＡ」はヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（参考例４で調製したポリクローナル抗体）における結果である。
【０１９８】
そして、この図において、「１」のバンドはヒト血清に含まれていたヒトＨＭＧ−１によるバンドであり、「２」のバンドはヒト血清に含まれていたヒトＨＭＧ−２によるバンドである。
〔これは、別途、ヒト血清の替わりに、精製して得たウシＨＭＧ−１及びウシＨＭＧ−２をそれぞれ電気泳動して、ウエスタンブロット法を行い、得られたバンドの位置より確かめておいた。（ヒトＨＭＧ−１とウシＨＭＧ−１、そしてヒトＨＭＧ−２とウシＨＭＧ−２のアミノ酸配列の相同性は、それぞれ非常に高いので、電気泳動においてほぼ同じ位置に泳動される。）〕
【０１９９】
（２）対照（コントロール）
前記１の（１）、（２）及び（３）における（１）の▲５▼の操作までを行ったもう一枚の各ニトロセルロース膜について、前記（１）の▲６▼の操作は行わず、しかし前記（１）の▲７▼以下の操作は同様に行って、これを対照（コントロール）とした。
【０２００】
この、ヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体も、ヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体をも作用させていない対照（コントロール）においては、ヒトＨＭＧ−１のバンドが現れる位置及びヒトＨＭＧ−２のバンドが現れる位置のいずれにおいても何ら発色は認められなかった。
このことより、前記の各ウエスタンブロット法においては、非特異的な発色が起きていないことが確かめられた。
【０２０１】
３．まとめ
図４より、実施例４で調製したヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（モノクローナル抗体）、及び参考例４で調製したヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（ポリクローナル抗体）では、ヒトＨＭＧ−１が泳動される位置とヒトＨＭＧ−２が泳動される位置の両方において発色が見られることが分かる。
このことより、ヒト血清中には、ヒトＨＭＧ−１とヒトＨＭＧ−２の両方が存在することが確かめられた。
【０２０２】
そして、前記のヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（モノクローナル抗体）とヒトＨＭＧ−１とヒトＨＭＧ−２の両方に結合する抗体（ポリクローナル抗体）は、確かにヒトＨＭＧ−１及びヒトＨＭＧ−２の両方に結合することが、このウエスタンブロット法の結果より確認できた。
【０２０３】
また、図４より、実施例４で調製したヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）では、ヒトＨＭＧ−１が泳動される位置には発色が見られるものの、ヒトＨＭＧ−２が泳動される位置には発色が見られないことが分かる。
【０２０４】
このことより、前記のヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体は、確かにヒトＨＭＧ−１には結合するものの、ヒトＨＭＧ−２には結合しないことが、このウエスタンブロット法の結果より確認できた。
【０２０５】
〔参考例１〕（ヒトＨＭＧ−１遺伝子の調製）
ヒトＨＭＧ−１をヒト脳ｃＤＮＡからＤＮＡ増幅を行いクローン化した。ＰＣＲ産物は、ＢａｍＨＩとＨｉｎｄIIIで修飾し、シークエンス・ベクターのｐＣＡＬ−ｎベクター（Ｓｔｒａｔａｇｅｎｅ社、カリフォルニア州、アメリカ合衆国）のＢａｍＨＩ−ＨｉｎｄIIIサイトにサブクローン化し、ＤＮＡ配列の確認を行った。
【０２０６】
〔参考例２〕（ＤＮＡ組換え法によるヒトＨＭＧ−１の調製）
このＢａｍＨＩ−ＨｉｎｄIIIで修飾されたＰＣＲ産物をグルタチオン−Ｓ−トランスフェラーゼとの融合タンパク質として発現させるために、ｐＥＸ発現ベクターのＢａｍＨＩ−ＨｉｎｄIIIサイトにサブクローン化した。
【０２０７】
次に、このリコンビナントプラスミドをＥ．ｃｏｌｉ・ＪＭ１に、トランスフォームした。
トランスフォームされた細胞を１Ｌ程で培養を行った後、ＩＰＴＧの誘導をかけ、グルタチオン−Ｓ−トランスフェラーゼ・ヒトＨＭＧ−１融合タンパク質を、Ｅ．ｃｏｌｉ・ＪＭ１で発現させた。
【０２０８】
この発現させたＥ．ｃｏｌｉ・ＪＭ１を集菌後、３０ｍＬ程度のリン酸緩衝生理食塩水（ＰＢＳ）〔１３７ｍＭ塩化ナトリウム、２．６８ｍＭ塩化カリウム、１．４７ｍＭリン酸二水素カリウム及び５．５９ｍＭリン酸水素二ナトリウムを含む水溶液（ｐＨ７．２）〕に分散させた。
【０２０９】
そして、これを超音波処理により破砕し、遠心分離後、上澄み液を回収した。
この上澄み液中に含まれるグルタチオン−Ｓ−トランスフェラーゼ・ヒトＨＭＧ−１融合タンパク質をグルタチオン・カラム（ファルマシア社）により精製した。
【０２１０】
次に、この精製したグルタチオン−Ｓ−トランスフェラーゼ・ヒトＨＭＧ−１融合タンパク質から、第Ｘａ因子を作用させて、ヒトＨＭＧ−１を切り出した。
以上のＤＮＡ組換え操作により、ヒトＨＭＧ−１を取得、調製した。
【０２１１】
〔参考例３〕（ウシＨＭＧ−１及びウシＨＭＧ−２の調製）
ウシの胸腺より、ウシＨＭＧ−１及びウシＨＭＧ−２をサンダースらの方法〔Ｃ．Ｓａｎｄｅｒｓら，Ｂ．Ｂ．Ｒ．Ｃ．，７８巻，１０３４〜１０４２頁，１９７７年発行〕に従って調製した。
▲１▼ まず、ウシの胸腺５００ｇを、１４０ｍＭの塩化ナトリウム及び０．５ｍＭのＰＭＳＦを含む６００ｍＬの緩衝液中で破砕を行った。
▲２▼ 次に、この破砕物を遠心分離機で遠心分離を行い、その上澄み液を除去した。
▲３▼ これに、１４０ｍＭの塩化ナトリウム及び０．５ｍＭのＰＭＳＦを含む緩衝液を加えて撹拌した後、遠心分離機で遠心分離を行い、その上澄み液を除去した。この洗浄操作を２回繰り返して行った。
▲４▼ 次に、得られた沈殿物に、０．７５Ｍの過塩素酸の３００ｍＬを加えた。そして、遠心分離機で遠心分離した後、上澄み液を分取した。残った沈殿物に０．７５Ｍの過塩素酸の４００ｍＬを加えた。これについても、遠心分離機で遠心分離した後、上澄み液を分取した。この上澄み液と先に分取した上澄み液とを合わせた。なお、沈殿物は廃棄した。
▲５▼ 前記の合わせた上澄み液に０．７５Ｍの過塩素酸を加えて、全体の容量を１，０００ｍＬとした。次に、遠心分離機で遠心分離した後、上澄み液をグラスフィルター（グレード４）で濾過した。
▲６▼ 前記の濾過の濾液に、３，５００ｍＬのアセトンと２１ｍＬの濃塩酸の混合液を加えた。濁りが生じてくるので、遠心分離機で遠心分離して、上澄み液を分取した。この上澄み液に、アセトン２，５００ｍＬを加えた。そして、再度、濁りが生じてくるので、これを遠心分離機で遠心分離して、上澄み液を分離し、残った沈殿物を集めた。
▲７▼ この集めた沈殿物を室温で自然乾燥させた。
【０２１２】
以上の操作により、ＨＭＧ−１及びＨＭＧ−２を含むタンパク質画分が、およそ２００ｍｇ得られた。
▲８▼ 前記のＨＭＧ−１及びＨＭＧ−２を含むタンパク質画分を、２００ｍＭ塩化ナトリウムを含む７．５ｍＭホウ酸ナトリウム緩衝液（ｐＨ９．０）の１０ｍＬに溶解した後、この２００ｍＭ塩化ナトリウムを含む７．５ｍＭホウ酸ナトリウム緩衝液（ｐＨ９．０）で充分に透析を行った。
▲９▼ この透析の後、７．５ｍＭホウ酸ナトリウム緩衝液（ｐＨ９．０）で平衡化しておいたＣＭ−セファデックスＣ２５のカラムに添加した。
【０２１３】
そしてその後、２００ｍＭ塩化ナトリウムを含む７．５ｍＭホウ酸ナトリウム緩衝液（ｐＨ９．０）により溶出させて、陽イオン交換クロマトグラフィーを行った。
この溶出パターンを図５に示した。なお、この図において、縦軸は２８０ｎｍにおける吸光度を示し、横軸は溶出画分の番号を示す。
【０２１４】
[10] そして、２０％ＳＤＳ−ポリアクリルアミド電気泳動の結果、その易動度より、図５において「Ａ」で示した溶出画分及び「Ｂ」で示した溶出画分はウシＨＭＧ−１を含む画分であり、更に「Ｃ」で示した溶出画分及び「Ｄ」で示した溶出画分はウシＨＭＧ−２を含む画分であることが確かめられた。
【０２１５】
よって、図５において「Ａ」で示した溶出画分及び「Ｂ」で示した溶出画分を混合して集め、更に「Ｃ」で示した溶出画分及び「Ｄ」で示した溶出画分を混合して集めた。
【０２１６】
〔参考例４〕（ポリクローナル抗体の調製）
参考例３で調製したウシＨＭＧ−１を用いてポリクローナル抗体の調製を下記のようにして行った。
〔１〕 動物への免疫
（１） 前記の参考例３で得たウシＨＭＧ−１を４００μｇ／ｍＬになるように生理食塩水（０．９％塩化ナトリウム水溶液）で溶解し、これをフロイント完全アジュバントと等量ずつ混合してエマルジョンとして、ウサギ（北山ラベス社）の腹部皮下に０．５ｍＬを免疫注射した。
【０２１７】
（２） 初回免疫から２週間後に、前記の免疫原を３００μｇ／ｍＬになるように生理食塩水で溶解し、これをフロイント不完全アジュバントと等量ずつ混合してエマルジョンとして、その０．５ｍＬにより追加免疫注射を行った。この追加免疫注射は２週間おきに行った。
【０２１８】
（３） 免疫動物であるこのウサギの血清中の抗体価を、酵素免疫測定法（ＥＬＩＳＡ、ＥＩＡ）にて、初回免疫から６週間目より１週間ごとに測定した。このＥＬＩＳＡ法の操作を以下に示した。
▲１▼ 参考例３で得たウシＨＭＧ−１を５μｇ／ｍＬになるように生理食塩水に溶解し、これを９６ウェル−マイクロプレート（ヌンク社製）に１ウェル当り１００μＬずつ加え、３７℃で２時間静置してこのウシＨＭＧ−１の固相化を行った。
▲２▼ このマイクロプレートを洗浄液（０．０５％ツイーン２０（Ｔｗｅｅｎ２０）を含むリン酸緩衝生理食塩水（５．５９ｍＭリン酸水素二ナトリウム、１．４７ｍＭリン酸二水素カリウム、１３７ｍＭ塩化ナトリウム及び２．６８ｍＭ塩化カリウムを含む水溶液（ｐＨ７．２）））で洗浄した後、１％ＢＳＡを含む１０ｍＭリン酸二水素カリウム−リン酸水素二カリウム緩衝液（ｐＨ７．２）を１ウェル当り３００μＬずつ加えて、３７℃で２時間静置してブロッキングを行い、その後再び洗浄液で洗浄した。
▲３▼ 抗体の産生を検査すべき前記ウサギの血清を、生理食塩水で１，０００倍、１０，０００倍、そして１００，０００倍と希釈し、これらをマイクロプレートのウェルに１００μＬずつ加え、３７℃で２時間静置して反応を行わせ、その後洗浄液で洗浄した。
▲４▼ また対照として、前記▲２▼のマイクロプレートのウェルに、１％ＢＳＡを含む０．１Ｍリン酸緩衝生理食塩水を１００μＬずつ加え、３７℃で２時間静置して、その後洗浄液で洗浄した。
▲５▼ パーオキシダーゼ（ＰＯＤ）標識抗ウサギＩｇＧ抗体（アマシャム社製）を３％ＢＳＡを含むリン酸緩衝生理食塩水で５，０００倍に希釈した後、▲３▼及び▲４▼のマイクロプレートに１ウェル当り１００μＬずつ加え、３７℃で２時間静置して反応を行わせた。
▲６▼ これを洗浄液で洗浄した後、パーオキシダーゼ反応液（３ｍＭ ２，２’−アジノ−ビス（３−エチルベンズチアゾリン−６−スルホン酸）〔ＡＢＴＳ〕を含む５０ｍＭリン酸水素二ナトリウム−２４ｍＭクエン酸緩衝液の１ｍＬに対して２μＬの１．７％過酸化水素を使用直前に添加したもの）を１ウェル当り１００μＬずつ加え、室温で反応させた。１５分後に１ウェル当り５０μＬの６Ｎ硫酸を加えて反応を停止させた。
▲７▼ これをＥＩＡプレートリーダー（バイオラッド社製）にて４１５ｎｍにおける吸光度の測定を行った。
【０２１９】
（４） 初回免疫から１２週間目以降、抗体価がプラトーに達したと認められたので、この免疫動物であるウサギの全採血を行った。そして、このウサギの血液より抗血清を得た。
（５） 前記（４）で得た抗血清について、２２〜２５℃において、この抗血清１ｍＬに対して０．１８ｇの比率で硫酸ナトリウムを撹拌しながら加え、硫酸ナトリウムが完全に溶けてから更に３０分間撹拌を続けて塩析を行った。
（６） これを２２〜２５℃で、遠心分離（７０００ｇ、１５分間）を行い、上澄み液と分離して得た沈殿をリン酸緩衝生理食塩水の１ｍＬに溶解した。
（７） 次に、これをリン酸緩衝生理食塩水に対して充分に透析した後、１０００ｇで２０分間遠心分離し不溶性のものを除去した。
（８） これをリン酸緩衝生理食塩水で平衡化しておいたＤＥＡＥ−セルロースイオン交換カラム（セルバ社製）〔１×２．５ｃｍ〕に通して、溶出液を集めた。
（９） 免疫グロブリンＧ（ＩｇＧ）が溶出液の素通り画分に含まれていることを２８０ｎｍの吸光度より確認し、これを集めて濃縮した。
【０２２０】
（１０） 次に、これを参考例２で調製したヒトＨＭＧ−１を固定化したカラムに通して、アフィニティークロマトグラフィーを行った。この操作を以下に示した。
▲１▼ 参考例２で調製したヒトＨＭＧ−１の２ｍｇに対して１ｇのＣＮＢｒ−セファロース（ファルマシアバイオテック社製）をその取扱説明書に従って反応させ、前記のＨＭＧ−１を固定化したアフィニティークロマトグラフィー用のカラムを調製した。
▲２▼ このカラムをリン酸緩衝生理食塩水で平衡化しておき、その後、前記（９）にて濃縮した成分（ポリクローナル抗体）を通した。
▲３▼ これにリン酸緩衝生理食塩水を充分に通して洗浄した後、０．１Ｍの酢酸緩衝液（ｐＨ３．０）を通した。
▲４▼ これにより溶出した画分を集め、リン酸緩衝生理食塩水で透析を行い、その後、濃縮を行った。
【０２２１】
以上のアフィニティークロマトグラフィーの操作により、ヒトＨＭＧ−１に結合するポリクローナル抗体を分取した。
【０２２２】
（１１） 以上の操作により得られたウサギのポリクローナル抗体は、ヒトＨＭＧ−１に結合することができるものである。また、ヒトＨＭＧ−１と相同性の高いヒトＨＭＧ−２とも結合することができるものである。
【０２２３】
〔実施例１０〕（抗体のヒトＨＭＧ−１及びヒトＨＭＧ−２との反応性の確認）
実施例４で調製したヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）について、ヒトＨＭＧ−１及びヒトＨＭＧ−２の各々との反応性をウエスタンブロット法により確かめた。
【０２２４】
なお、前記実施例９においては、ヒト血清に含まれていたヒトＨＭＧ−１及びヒトＨＭＧ−２と各抗体との反応性を確かめたが、本実施例においては、ヒト細胞より調製したヒトＨＭＧ−１及びヒトＨＭＧ−２と前記抗体との反応性の確認を行った。
【０２２５】
１．ウエスタンブロット法
（１）ヒトＨＭＧ−１及びヒトＨＭＧ−２を電気泳動して転写したものへ、ヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）を反応させたウエスタンブロット法
▲１▼ 後述する参考例５においてヒト細胞（ＨＬ６０細胞）より調製したヒトＨＭＧ−１及びヒトＨＭＧ−２をタイタン・ジェル・リポタンパク質電気泳動キット（ヘレナ研究所社製）を用いて電気泳動を行った。なお、支持体はアガロースゲルであり、これに前記のヒトＨＭＧ−１及びヒトＨＭＧ−２を各々０．５ｍｇ／ｍＬとなるように溶解した２００ｍＭ塩化ナトリウムを含む７．５ｍＭホウ酸緩衝液（ｐＨ９．０）の２μＬを接触させた。
▲２▼ そして、泳動緩衝液としてバルビタール緩衝液（ｐＨ８．８）を使用して、電圧９０Ｖで７５分間通電して電気泳動を行った。なお、この電気泳動は、同じものを２セット用意して行い、以下の操作も同様に行った。
▲３▼ 前記▲２▼の電気泳動の後の転写は、ノバ・ブロット・エレクトロフォレティック・トランスファー・キット（ファルマシア−エルケービー社製）を用いて、その使用説明書に従い、ドライ方式で行った。
【０２２６】
まず、前記▲２▼において電気泳動を行ったアガロースゲルを転写用装置上に置いた。次に、このアガロースゲルの上に、９ｃｍ×９ｃｍのニトロセルロース膜（バイオラッド社製）を重ね、４８ｍＭトリス（ヒドロキシメチル）アミノメタン、３９ｍＭグリシン、０．０３７５％（Ｗ／Ｖ）ドデシル硫酸ナトリウム（ＳＤＳ）及び２０％（Ｖ／Ｖ）メタノールよりなる転写用緩衝液を用いて、電流６５ｍＡで２時間転写を行った。
【０２２７】
▲４▼ この転写を行ったニトロセルロース膜を、１％ＢＳＡを含むリン酸緩衝生理食塩水（５．５９ｍＭリン酸水素二ナトリウム、１．４７ｍＭリン酸二水素カリウム、１３７ｍＭ塩化ナトリウム及び２．６８ｍＭ塩化カリウムを含む水溶液（ｐＨ７．２））の２０ｍＬに４℃で１晩浸漬して、ブロッキングを行った。
▲５▼ 次に、これを洗浄液（０．０５％ツイーン２０（Ｔｗｅｅｎ２０）を含むリン酸緩衝生理食塩水）の２０ｍＬ中で１０分間振とう洗浄を行った。この操作を３回行った。
▲６▼ 実施例４で調製した、ヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）を２０ｍＬのリン酸緩衝生理食塩水に８０μｇ溶解し、この溶液に前記▲５▼の操作を行ったニトロセルロース膜を室温で２時間浸漬して反応させた。
▲７▼ 前記▲６▼の操作を行ったニトロセルロース膜を、２０ｍＬの洗浄液中で１０分間振とう洗浄を行った。これを３回行った。
▲８▼ 次に、パーオキシダーゼ標識抗マウスＩｇＧ抗体（ダコ社製）を、３％ＢＳＡを含むリン酸緩衝生理食塩水で５００倍希釈をして、２０ｍＬの溶液を調製し、これに前記▲７▼のニトロセルロース膜を室温で２時間浸漬して反応させた。
▲９▼ このニトロセルロース膜を、２０ｍＬの洗浄液中で１０分間振とう洗浄を行った。この操作を３回行った。
[10] ０．０２５％の３，３’−ジアミノベンジジン四塩酸塩及び０．０１％過酸化水素を含むリン酸緩衝生理食塩水の２０ｍＬに、前記▲９▼のニトロセルロース膜を室温で１５分間浸漬して発色させた。
【０２２８】
以上の操作により、実施例４で調製した、ヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）の、ヒトＨＭＧ−１及びヒトＨＭＧ−２との反応性に関するウエスタンブロット法の結果を得た。
【０２２９】
（２）ヒトＨＭＧ−１及びヒトＨＭＧ−２を電気泳動して転写したものへ、ヒトＨＭＧ−２に結合する抗体（ポリクローナル抗体）を反応させたウエスタンブロット法
参考として、前記のヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）に替えて、市販のヒトＨＭＧ−２に結合する抗体（ポリクローナル抗体）〔品名：Ａｎｔｉ ＨＭＧ−２（Ｃ−１９），Human、品番：SC-8758、メーカー：サンタクルズ社（米国）、販売：コスモ・バイオ社〕を前記（１）の▲６▼において用いること以外は、前記（１）の▲１▼〜[10]の通りに操作を行い、バンドを得て、本ウエスタンブロット法におけるヒトＨＭＧ−２のバンドの位置の確認に用いた。
【０２３０】
（３）ウシＨＭＧ−１及びウシＨＭＧ−２を電気泳動して転写したものへ、ヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−２には結合しない抗体（モノクローナル抗体）を反応させたウエスタンブロット法
参考として、前記のヒトＨＭＧ−１及びヒトＨＭＧ−２に替えて、参考例３においてウシ胸腺より調製したウシＨＭＧ−１及びウシＨＭＧ−２の等量混合液を用いること以外は、前記（１）の▲１▼〜[10]の通りに操作を行い、バンドを得て、本ウエスタンブロット法におけるウシＨＭＧ−１のバンドの位置の確認に用いた。
【０２３１】
２．実験結果
（１）ウエスタンブロット法の結果
前記１におけるウエスタンブロット法の結果を図６に示した。
なお、この図において、「Ｈ▲１▼」のレーンは前記１の（１）のウエスタンブロット法のレーンであり、「Ｈ▲２▼」のレーンは前記１の（２）のウエスタンブロット法のレーンであり、そして、「Ｂ▲１▼」のレーンは前記１の（３）のウエスタンブロット法のレーンである。
【０２３２】
この図６より、以下のことが分かる。
・ 「Ｈ▲１▼」のレーンにおいては、ただ一本のバンドだけが認められる。
・ 「Ｈ▲１▼」のレーンにおいては、「Ｈ▲２▼」のレーンにおけるバンドの位置にはバンドが認められない。
・ 「Ｈ▲１▼」のレーンと「Ｂ▲１▼」のレーンは、同じ位置にバンドが認められる。
【０２３３】
すなわち、
▲１▼ ヒトＨＭＧ−２に結合する抗体（ポリクローナル抗体）を反応させた「Ｈ▲２▼」のレーンのバンドの位置に、「Ｈ▲１▼」のレーンではバンドが認められないことが分かる。
▲２▼ ヒト細胞より調製したヒトＨＭＧ−１及びヒトＨＭＧ−２を泳動させた「Ｈ▲１▼」のレーンにおいて、ただ一本のバンドだけが認められ、そのバンドの位置が、ヒトＨＭＧ−１と非常に相同性の高いウシＨＭＧ−１及びヒトＨＭＧ−２と非常に相同性の高いウシＨＭＧ−２を泳動させた「Ｂ▲１▼」のレーンの唯一のバンドの位置と同じであることが分かる。
【０２３４】
以上のことより、実施例４で調製された抗体（モノクローナル抗体）は、ヒト細胞より調製したヒトＨＭＧ−１に結合し、かつ、ヒト細胞より調製したヒトＨＭＧ−２には結合しないことが確かめられた。
【０２３５】
（２）対照（コントロール）
前記１の（１）の▲５▼の操作までを行ったもう一枚のニトロセルロース膜について、前記▲６▼の操作は行わず、しかし前記▲７▼以下の操作は同様に行って、これを対照（コントロール）とした。
前記１の（２）及び（３）においても、同様に対照（コントロール）を作成した。
【０２３６】
これらのいずれの抗体も作用させていない対照（コントロール）においては、何ら発色は認められなかった。
このことより、前記の各ウエスタンブロット法においては、非特異的な発色が起きていないことが確かめられた。
【０２３７】
〔参考例５〕（ヒト細胞よりのヒトＨＭＧ−１及びヒトＨＭＧ−２の調製）
ヒト細胞（ＨＬ６０細胞）より、ヒトＨＭＧ−１及びヒトＨＭＧ−２を調製した。
▲１▼ まず、ＲＰＭＩ１６４０にて培養したヒト細胞（ＨＬ６０細胞）の培養液の上清の３Ｌを、約２５０ｍＬに濃縮した。
▲２▼ 次に、終濃度が２００ｍＭとなるように、塩化ナトリウムを添加した。
▲３▼ これを遠心分離機で遠心分離を行い（１０，０００ｒｐｍ、３０分間）、その上清を分取して、ポアサイズ０．４５μｍのフィルターで濾過を行った。
▲４▼ この濾液を、ハイトラップ・ヘパリン・カラム（アマシャムファルマシア社製）に通した。
▲５▼ このカラムに、２００ｍＭ塩化ナトリウムを含む１０ｍＭリン酸ナトリウム緩衝液（ｐＨ７．０）を流して洗った。
▲６▼ 次に、リン酸ナトリウム緩衝液（ｐＨ７．０）において、塩化ナトリウム濃度が２００ｍＭから２，０００ｍＭまでのグラジエントをかけて前記カラムより溶出させた。
▲７▼ 前記の各溶出画分をＳＤＳ−ポリアクリルアミド電気泳動にかけ、その易動度よりヒトＨＭＧ−１及びヒトＨＭＧ−２を含む画分を同定した。この画分は、塩化ナトリウム濃度が５００ｍＭから１，０００ｍＭにあるときに溶出した画分であった。
▲８▼ 前記▲７▼のヒトＨＭＧ−１及びヒトＨＭＧ−２を含む画分を、７．５ｍＭホウ酸ナトリウム緩衝液（ｐＨ９．０）で平衡化しておいたＣＭ−セファデックスＣ２５のカラムに通した。
そしてその後、２００ｍＭ塩化ナトリウムを含む７．５ｍＭホウ酸ナトリウム緩衝液（ｐＨ９．０）により溶出させて、陽イオン交換クロマトグラフィーを行った。
▲９▼ ここで溶出した各画分を、ＳＤＳ−ポリアクリルアミド電気泳動にかけ、その易動度よりヒトＨＭＧ−１を含む画分、及びヒトＨＭＧ−２を含む画分を各々同定した。
以上の操作により、ヒト細胞（ＨＬ６０細胞）より、ヒトＨＭＧ−１を調製し、また、ヒトＨＭＧ−２をも調製した
【０２３８】
【発明の効果】
本発明の抗体は、ヒトＨＭＧ−１には結合するが、ヒトＨＭＧ−１と非常に相同性の高いヒトＨＭＧ−２には結合しない抗体である。
【０２３９】
また、本発明のヒトＨＭＧ−１の免疫学的測定試薬は、ＨＭＧ−２を測り込むことがなく、ＨＭＧ−１だけを測定することができ、かつ測定の操作が簡便で自動化することも可能な測定試薬である。これにより、誤差を含まない正確なＨＭＧ−１の測定値を得ることができ、敗血症等の疾患の診断において、診断を誤らせることを防ぐことができるものである。
【０２４０】
そして、本発明のヒトＨＭＧ−１の免疫学的測定方法は、ＨＭＧ−２を測り込むことがなく、ＨＭＧ−１だけを測定することができ、かつ測定の操作が簡便で自動化することも可能な測定方法である。これにより、誤差を含まない正確なＨＭＧ−１の測定値を得ることができ、敗血症等の疾患の診断において、診断を誤らせることを防ぐことができるものである。
【０２４１】
【配列表】

【０２４２】
【配列表フリーテキスト】
配列番号１：ヒトＨＭＧ−１のアミノ酸配列に基づいて合成したペプチド
配列番号２：ヒトＨＭＧ−１のアミノ酸配列に基づいて合成したペプチド
【図面の簡単な説明】
【図１】ヒトＨＭＧ−１のアミノ酸配列の全てについて、ホップらの方法により、各アミノ酸残基の親水性の高さの推定を行った結果を示した図である。
【図２】本発明の免疫学的測定試薬、及び免疫学的測定方法により、ヒトＨＭＧ−１を含む試料を測定して作成した検量線を示した図である。
【図３】本発明の免疫学的測定試薬、及び免疫学的測定方法により、ウシＨＭＧ−１を含む試料を測定して作成した検量線を示した図である。
【図４】各々の抗体の、ヒトＨＭＧ−１及びヒトＨＭＧ−２との反応性を確かめたウエスタンブロット法の結果を示した図である。
【図５】ウシＨＭＧ−１及びウシＨＭＧ−２の調製における陽イオン交換クロマトグラフィーの溶出パターンを示した図である。
【図６】各々の抗体の、ヒトＨＭＧ−１及びヒトＨＭＧ−２との反応性を確かめたウエスタンブロット法の結果を示した図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an antibody that specifically binds to human high mobility group protein-1 that can be a marker for diseases such as sepsis, and an immunoassay reagent and immunity for human high mobility group protein-1 using this antibody. It is related to the method of measuring.
[0002]
The present invention is useful in the field of life science such as clinical examination, clinical pathology, immunology and medicine, and in the field of chemistry such as analytical chemistry.
[0003]
[Prior art]
High Mobility Group Protein (hereinafter sometimes abbreviated as “HMG”) was discovered in 1964 as a large amount of non-histone protein contained in chromatin structure and is universally included in all higher animals and plants. It has a very high degree of conservation of primary structure among tribes. It has also been found that it exists not only in the nucleus but also in the cytoplasm. Although the physiological effect is not clearly understood, HMG relaxes the double helix structure when binding to DNA, so that the transcriptional activity is increased by changing the higher order structure of DNA to the optimal structure during the transcription reaction. It is thought to function as a very wide range of transcriptional promoters and nucleosome relaxing factors.
[0004]
There are several types of HMG. For example, high mobility group protein-1 (HMG-1), high mobility group protein-2 (HMG-2), high mobility group protein-3 (HMG-3), high mobility group protein-8 (HMG-8), High Mobility Group Protein-17 (HMG-17), High Mobility Group Protein-I (HMG-I), High Mobility Group Protein-Y (HMG-Y), High Mobility Group Protein-I (Y) (HMG-I ( Y)), high mobility group protein IC (HMG IC) and the like.
[0005]
In addition, when the present inventors analyzed the homology of amino acid sequences using genetic information processing software “GENETYX” (Software Development), bovine HMG-1 was compared with human HMG-1. Was 98.6% and porcine HMG-1 homology was 99.1%. Similarly, human HMG-2 has a homology of 81.2%, bovine HMG-2 has a homology of 72.3%, and porcine HMG-. The homology of 2 was 79.4%.
[0006]
In 1999, Wang et al. Quantitatively measured HMG-1 in serum (in blood) for the first time by Western blotting using a polyclonal antibody prepared using HMG-1 itself as an immunogen. As a result, Wang et al. Showed that HMG-1 can be a marker for sepsis. And, it was shown that it is possible to accurately measure HMG-1 in blood in order to discriminate between surviving patients and patients dying among septic patients. That is, the usefulness of not only confirming the presence of HMG-1 in blood but also precise quantification was revealed (H. Wang et al., SCIENCE, Vol. 285, No. 9, 248-251). Page, 1999).
[0007]
It has been previously shown that antibodies used for the measurement of HMG-1, that is, antibodies that bind to HMG-1, can be prepared by Parkinen et al. And Rep et al. (J. Parkkinen et al., The Journal of Biological Chemistry, 268, 26, 19726-19738, published in 1993; WA Lepp et al., Journal of Immunoassay, 10, 4, 449-465, published in 1989). Using this antibody, Rep et al. State that Solid-phase Enzyme Immunoassay is possible for HMG-1. (In this solid-phase enzyme immunoassay, purified HMG-1 is immobilized on a well of a microplate (microtiter plate), and an antibody that binds to enzyme-labeled HMG-1 is brought into contact therewith to act. It was confirmed that the antibody that binds to HMG-1 binds to purified HMG-1.)
However, since these antibodies are polyclonal antibodies prepared using HMG-1 itself as an immunogen, its amino acid sequence is highly homologous to HMG-1 (81.2%) and binds to HMG-2. It was an end.
[0008]
That is, the measuring method of HMG-1 shown by Rep et al. Or Wang et al. Was a measuring method that measured not only HMG-1 but also HMG-2.
[0009]
In 2000, Luhiainen et al. Also developed a polyclonal antibody prepared by using genetically engineered rat HMG-1 itself as an immunogen and a part of the amino acid sequence of HMG-1 “Lys Phe Lys Asp Pro Asn”. Using each of the polyclonal antibodies prepared using the peptide consisting of “Ala Pro Lys Arg Pro Pro Ser Ala” as an immunogen, HMG-1 in human blood was measured by an ELISA sandwich method (A. Rouhiainen et al., Thromb). Haemost, 84, 1087-1094, 2000).
[0010]
However, the polyclonal antibody prepared by using rat HMG-1 itself as an immunogen used here binds not only to HMG-1 but also to HMG-2 for the reasons described above.
[0011]
In addition, a polyclonal antibody prepared by using a peptide consisting of a part of the amino acid sequence of HMG-1 “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” as an immunogen is an amino acid sequence of the peptide used as an immunogen. However, since it contains a part of the amino acid sequence of HMG-2 and is a polyclonal antibody, it also binds not only to HMG-1 but also to HMG-2.
[0012]
Therefore, the measurement method disclosed by Ruhiainen et al. Is also a measurement method that measures not only HMG-1 but also HMG-2.
[0013]
Thus, until now, there has been no known antibody that binds to HMG-1 but does not bind to HMG-2.
[0014]
Therefore, there is no measurement method capable of measuring only HMG-1 without measuring HMG-2 other than the measurement method by the Western blot method of Wang et al. (In this Western blot method, HMG-1, HMG-2, etc. are electrophoresed, the antibody is brought into contact with and bound to this carrier or a carrier to which this carrier has been transferred. Therefore, since the migration positions of HMG-1 and HMG-2 (mobility due to the difference in molecular weight) are different, HMG-1 and HMG-2 can be separated.)
[0015]
[Problems to be solved by the invention]
As described above, an antibody that binds to HMG-1 but does not bind to HMG-2 is not known and did not exist. For this reason, except for the method of Western blotting by Wang et al., An immunological measurement method that can measure only HMG-1 which can be a marker for diseases such as sepsis and does not measure HMG-2. There was no immunoassay reagent. That is, in immunological measurement methods and immunological measurement reagents other than Western blotting, even when trying to measure HMG-1 in a sample, HMG-2 contained in the sample is also measured, and therefore positive. A measurement value of HMG-1 including an error is obtained. This HMG-2 is also measured, and the measured value of HMG-1 including a positive error may cause a diagnosis of a disease such as sepsis to be mistaken.
[0016]
The Western blot method is a measurement method that is complicated in operation and requires skill in the technique, and therefore, the number of measurers and measurement facilities that can be implemented is limited. In the Western blot method, it is difficult to automate the measurement.
[0017]
Therefore, a measuring method and a measuring reagent that can accurately measure only HMG-1 without measuring HMG-2, and that are simple in operation or can be automated. Was demanded.
[0018]
On the other hand, the first object of the present invention is to provide an antibody that binds to human HMG-1 but does not bind to human HMG-2.
[0019]
In addition, the second problem of the present invention is that HMG-2 is not measured, an accurate measurement value of HMG-1 that does not include an error can be obtained, and the measurement operation is simple and can be automated. Another object is to provide an immunoassay reagent for human HMG-1.
[0020]
The third problem of the present invention is that HMG-2 is not measured, an accurate measurement value of HMG-1 that does not include an error can be obtained, and the operation is simple and can be automated. It is to provide a method for immunological measurement of human HMG-1.
[0021]
[Means for Solving the Problems]
The present invention includes the following inventions.
(1) An antibody that binds to human high mobility group protein-1 but does not bind to human high mobility group protein-2.
(2) The following formula (I):
Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys (I)
The antibody according to (1) above, which is prepared by using a peptide consisting of the amino acid sequence represented by
(3) Features of (1) and (2) below:
(1) The following formula (I):
Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys (I)
Consisting of an amino acid sequence obtained by performing deletion, substitution, insertion or addition, or modification of one to several amino acid residues to the amino acid sequence represented by
(2) When an antibody is prepared using the peptide as an immunogen, an antibody that binds to human high mobility group protein-1 but does not bind to human high mobility group protein-2 can be obtained.
The antibody according to (1) above, which is prepared by using a peptide having the above as an immunogen.
(4) Features of (1) and (2) below:
(1) The following formula (I):
Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys (I)
Consisting of an amino acid sequence obtained by performing deletion, substitution, insertion or addition, or modification of one to several amino acid residues to the amino acid sequence represented by
(2) It can bind to an antibody prepared by using a peptide comprising the amino acid sequence represented by the formula (I) as an immunogen,
The antibody according to (1) above, which is prepared by using a peptide having the above as an immunogen.
(5) The antibody according to any one of (1) to (4), which is a monoclonal antibody.
(6) Use of the antibody according to any one of (1) to (5) above in an immunological measurement method for measuring human high mobility group protein-1 contained in a sample using an antigen-antibody reaction An immunological measurement method comprising:
(7) An immunoassay reagent for measuring human high mobility group protein-1 contained in a sample using an antigen-antibody reaction, wherein the antibody according to any one of (1) to (5) above An immunological measurement reagent comprising:
[0022]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
1. An antibody that binds to human HMG-1 but does not bind to human HMG-2
(1) General
An antibody that binds to human high mobility group protein-1 (human HMG-1) but does not bind to human high mobility group protein-2 (human HMG-2) of the present invention (hereinafter referred to as “antibody of the present invention”). May be any antibody as long as it binds to human HMG-1 and does not bind to human HMG-2.
[0023]
The antibodies of the present invention include polyclonal antibodies, antisera consisting of polyclonal antibodies, or monoclonal antibodies, and fragments of these antibodies (Fab, F (ab ′)). ₂ Or Fab ′ etc.).
The antibody of the present invention is preferably a monoclonal antibody because the absorption operation can be omitted in the preparation operation.
[0024]
The method for obtaining the antibody of the present invention is not particularly limited, and examples thereof include the following methods.
(1) The amino acid sequence of HMG-1 is compared with the amino acid sequence of HMG-2, and the amino acid sequence of HMG-1 having low homology between HMG-1 and HMG-2 is selected. Next, a peptide or protein containing this amino acid sequence is prepared and obtained, and this is used as an immunogen to immunize an animal to obtain the produced antibody. Then, it is confirmed that the antibody binds to human HMG-1 and does not bind to human HMG-2, or such an antibody is selected to obtain the antibody of the present invention.
[0025]
The amino acid sequence is preferably a highly hydrophilic portion. This is because the higher the hydrophilicity, the higher the possibility that the amino acid sequence is present on the surface of the HMG-1 molecule, and therefore the higher the possibility that an antibody produced using this as an immunogen can bind to HMG-1. .
[0026]
(2) A peptide or protein containing all or part of the amino acid sequence of HMG-1 is prepared and obtained, and the animal is immunized with this as an immunogen to obtain the produced antibody. A solution containing the produced antibody and HMG-2 immobilized on a solid phase is brought into contact, and an antibody that binds to HMG-2 is bound to the solid phase via the immobilized HMG-2. Next, by separating the carrier and the solution, an antibody that binds to HMG-2 is removed from the solution, and an antibody that binds to human HMG-1 and does not bind to human HMG-2 is obtained.
[0027]
(3) The amino acid sequence of HMG-1 is compared with that of HMG-2, and the amino acid sequence of HMG-1 having low homology between HMG-1 and HMG-2 is selected. Next, a peptide or protein containing this amino acid sequence is prepared and obtained, and this is used as an immunogen to immunize an animal to obtain the produced antibody. A solution containing the produced antibody and HMG-2 immobilized on a solid phase is brought into contact, and an antibody that binds to HMG-2 is bound to the solid phase via the immobilized HMG-2. Next, by separating the carrier and the solution, an antibody that binds to HMG-2 is removed from the solution, and an antibody that binds to human HMG-1 and does not bind to human HMG-2 is obtained.
[0028]
In this case as well, the amino acid sequence is preferably a highly hydrophilic portion. This is because the higher the hydrophilicity, the higher the possibility that the amino acid sequence is present on the surface of the HMG-1 molecule, and therefore the higher the possibility that an antibody produced using this as an immunogen can bind to HMG-1. .
[0029]
The method (1) is suitable for obtaining monoclonal antibodies, and the methods (2) and (3) are suitable for obtaining polyclonal antibodies or antisera. The method (2) is more complicated to operate than the method (1), and the method (3) is more complicated to operate than the method (2). Since it is complicated, the method (1) is more preferable from the viewpoint of operation.
[0030]
(2) Immunogen
The immunogen for producing the antibody of the present invention will be described below.
As described above, as an immunogen for producing the antibody of the present invention, a peptide or protein containing all or part of the amino acid sequence of HMG-1, or low homology between HMG-1 and HMG-2 A peptide or protein containing the amino acid sequence of HMG-1 can be used.
[0031]
The amino acid sequence is preferably a highly hydrophilic portion. This is because the higher the hydrophilicity, the higher the possibility that the amino acid sequence is present on the surface of the HMG-1 molecule, and therefore the higher the possibility that an antibody produced using this as an immunogen can bind to HMG-1. .
[0032]
The estimation of the hydrophilicity of each amino acid residue of this amino acid sequence is carried out by the method of Hop et al. (TP Hopp et al., Proc. Natl. Acad. Sci. USA, 78, 3824- 3828, published in 1981) or by Parker et al. (Parker et al., Biochemistry, Vol. 25, pages 5425-5432, published in 1986).
[0033]
The inventors of the present invention have described the amino acid sequence of human HMG-1 (L. Wen et al., Nucleic Acids Res., 17, pp. 1197-1214, published in 1989) according to the method of Hop et al. The estimation of the hydrophilicity of the residue was performed. The results are shown in FIG.
[0034]
In this figure, the horizontal axis indicates the order from the N-terminus of each amino acid residue of human HMG-1. On the other hand, the vertical axis indicates that the higher the value is on the positive side, the higher the hydrophobicity is. On the contrary, the higher the value is on the negative side, the higher the hydrophilicity is.
[0035]
Next, the present inventors converted the highly hydrophilic sequence of the amino acid sequence of human HMG-1 into the amino acid sequence of human HMG-2 (M. Yoshida et al., J. Biol. Chem., 267, 6641-1). 6645, published in 1992).
Then, from this highly hydrophilic amino acid sequence, the amino acid sequence of human HMG-1 having low homology between human HMG-1 and human HMG-2 was selected.
[0036]
The amino acid sequence selected by the present inventors is “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly from the N-terminal amino acid residue (glycine) to the 11th amino acid residue (lysine) of human HMG-1. Lys ".
[0037]
As will be described later, an antibody prepared using a peptide consisting of this amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” as an immunogen binds to human HMG-1, but does not bind to human HMG-2. An antibody that does not bind (an antibody of the present invention).
[0038]
The antibody of the present invention can be obtained by preparing as an immunogen a peptide consisting of the amino acid sequence represented by this “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys”. As an immunogen for producing glycine, 1 to several amino acids (usually 1 to 8, preferably 1 to 6) in the amino acid sequence represented by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” It may be a peptide consisting of an amino acid sequence obtained by deletion, substitution, insertion or addition, or modification of a residue.
[0039]
The amino acid sequence of human HMG-2 corresponding to the amino acid sequence of human HMG-1 is “Gly Lys Gly Asp Pro Asn Lys Pro Arg Gly Lys”, which is the sixth amino acid residue from the N-terminal “ Since “Lys” (HMG-1) is simply replaced by “Asn” (HMG-2), and the only difference in the amino acid sequence between human HMG-1 and human HMG-2 is this point, human HMG Among the immunogens of an antibody that binds to -1 but does not bind to human HMG-2 (the antibody of the present invention), an immunogen derived from the amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” It is considered essential to include the sixth amino acid residue “Lys” from the N-terminal of human HMG-1.
[0040]
Since there is a report that an antibody can recognize an amino acid sequence consisting of three amino acids (F. Hudecz et al., J. Immunol. Methods, 147, 201-210, published in 1992), Among antibody immunogens, “Pro Lys Lys”, “Asp Pro Lys”, or “Lys Lys Pro” can be defined (considered) as the minimum unit of an immunogen derived from the amino acid sequence. Furthermore, those obtained by adding other amino acids or peptides to these tripeptides can be considered as immunogens of the antibody of the present invention.
[0041]
Deletion of one to several amino acid residues in the amino acid sequence represented by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” as an immunogen for producing the antibody of the present invention, A peptide comprising an amino acid sequence obtained by substitution, insertion or addition, or modification satisfies one of the following conditions.
(A) a peptide capable of obtaining an antibody that binds to human HMG-1 but does not bind to human HMG-2 when an antibody is prepared using the peptide as an immunogen, or
(B) A peptide capable of binding to an antibody prepared using a peptide having an amino acid sequence represented by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” as an immunogen.
[0042]
In addition, when following the method (2) of (1), a peptide or protein containing all or part of the amino acid sequence of HMG-1 can be prepared and obtained, and this can be used as an immunogen.
[0043]
As the aforementioned immunogen, a peptide consisting of an amino acid sequence represented by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys”, an amino acid sequence represented by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” A peptide comprising an amino acid sequence obtained by deletion, substitution, insertion or addition, or modification of one to several amino acid residues, or a peptide or protein comprising all or part of the amino acid sequence of HMG-1 It can be extracted and purified from body fluids, cells, tissues or organs of humans or other animals by a known method.
[0044]
The method for obtaining HMG-1 or HMG-2 from human thymus, porcine thymus, bovine thymus, human placenta, neutrophil, HL-60 cell line, etc. is described in the following documents (H Goodwin et al., Biochema Biophysica Acta, 405, 280-291, published in 1975; M. Yoshida et al., J. Biochem., 95, 117-124, published in 1980; 530, 39-46, 1992).
[0045]
Since a mixture of bovine HMG-1 and bovine HMG-2 is sold by Wako Pure Chemical Industries, only bovine HMG-1 can be purified and obtained.
[0046]
Each of the immunogens can be synthesized by a peptide synthesis method such as a liquid phase method and a solid phase method, and an automatic peptide synthesizer may be used. 1 “Chemistry of Protein IV”, Tokyo Kagaku Doujin, 1975, Izumiya et al. “Peptide Synthesis Fundamentals and Experiments”, Maruzen, 1985, “Sequence Chemistry Laboratory 2 Protein Chemistry” edited by Japanese Biochemical Society, Tokyo Kagaku Doujin , 1987, etc., and it is easy to produce a mutant in which the amino acid sequence is deleted, substituted, inserted or added. In addition, modifications such as introduction of unnatural amino acids, chemical modification of each amino acid residue, or cyclization of the interior of the molecule by introduction of cysteine residues to stabilize the structure may be performed.
[0047]
Furthermore, each of the immunogens may be prepared from DNA or RNA having a corresponding nucleobase sequence by using genetic engineering technology, edited by the Japanese Biochemical Society, “Sequential Biochemistry Experiment Course 1 Gene Research Method I”, Tokyo Chemical Doujin, 1986, Japan Biochemical Society, “Sequential Biochemistry Course 1, Genetic Research Method II”, Tokyo Chemical Doujin, 1986, Japan Biochemical Society, “Sequential Biochemistry Experimental Course 1, Genetic Research Method III”, Tokyo Chemical Doujin 1987 and the like.
[0048]
By the way, when the immunogen is a low molecular weight substance, it is common to immunize an animal or the like with a carrier bound to the immunogen. However, a specific antibody against the peptide having 5 amino acids is used as an immunogen. Since there is a report that it was produced (Kiyama et al., “Abstract 3 of the 112th Annual Meeting of the Japanese Pharmaceutical Society”, page 122, published in 1992), it is not essential to use a carrier.
[0049]
In addition, as a carrier when using a carrier when producing antibodies, mussel hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), chicken serum albumin, poly-L-lysine, poly Known carriers such as alanyl lysine, dipalmityl lysine, tetanus toxoid or polysaccharide can be used.
[0050]
The immunogen and carrier binding methods include glutaraldehyde method, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide method, maleimidobenzoyl-N-hydroxysuccinimide ester method, bisdiazotized pentidine method or N-succimidyl- A known binding method such as a 3- (2-pyridyldithio) propionic acid method can be used.
Moreover, what made the immunogen adsorb | suck to carriers, such as a nitrocellulose particle | grain, polyvinylpyrrolidone, or a liposome, can also be used as an immunogen.
[0051]
(3) Antibody acquisition method
(1) Polyclonal antibody, antiserum
In the antibody of the present invention, the polyclonal antibody or antiserum can be obtained by the following operation.
[0052]
First, a mammal (mouse, rabbit, rat, sheep, goat, horse, etc.) or a bird (chicken, etc.) is immunized with the immunogen or a conjugate of the immunogen and a carrier.
The amount of immunization of the immunogen or the conjugate of the immunogen and the carrier is determined by the type of immunized animal, the site of immunization, and the like. Each mouse is injected with 0.1 μg to several mg, preferably 50 μg to 1 mg of the immunogen or a conjugate of the immunogen and a carrier at a time. In the case of rabbits, 10 μg to several tens mg of the immunogen or a conjugate of the immunogen and a carrier is immunized once per rabbit.
[0053]
The immunogen or the combined immunogen and carrier is preferably added and mixed with an adjuvant for immunization injection. As the adjuvant, known ones such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant or pertussis adjuvant can be used.
[0054]
Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
After the initial immunization, booster injections of the immunogen or the conjugate of the immunogen and the carrier are given at sites such as subcutaneous, intravenous, intraperitoneal or back at intervals of 2 to 3 weeks. Also in this case, the immunogen or the conjugate of the immunogen and the carrier is preferably boosted by adding an adjuvant and mixing.
[0055]
After the initial immunization, the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the whole blood is collected, and the serum is separated to obtain an antiserum containing the antibody of the present invention. .
[0056]
The antiserum is subjected to antibody purification by a salting-out method using ammonium sulfate, sodium sulfate or the like, ion exchange chromatography, gel filtration method or affinity chromatography, or a combination of these methods to obtain a polyclonal antibody.
[0057]
The obtained polyclonal antibody is composed of both a polyclonal antibody that binds to HMG-1 but does not bind to HMG-2 and a polyclonal antibody that binds to both HMG-1 and HMG-2. Therefore, this is further passed through an affinity chromatography column immobilized with HMG-2 as a ligand on a solid phase. Polyclonal antibodies that bind to both HMG-1 and HMG-2 bind to the solid phase via the ligand (HMG-2) of this column and are collected. In contrast, a polyclonal antibody that binds to HMG-1 but does not bind to HMG-2 passes through this column without binding to the ligand (HMG-2) of this column. By obtaining the fraction, a polyclonal antibody that binds to human HMG-1 but does not bind to human HMG-2 can be obtained.
[0058]
In addition, when an animal or the like is immunized using a conjugate of an immunogen and a carrier, an antibody against this carrier exists in the obtained antiserum or polyclonal antibody. It is preferable to carry out. As this removal treatment method, a carrier is added to the obtained polyclonal antibody or antiserum solution to remove aggregates generated, or the carrier is immobilized on an insolubilized solid phase and removed by affinity chromatography, etc. Can be used.
[0059]
(2) Monoclonal antibody
In the antibody of the present invention, the monoclonal antibody can be obtained by the following operation.
The monoclonal antibody is an antibody-producing cell such as a hybridoma obtained by the cell fusion method of Keller et al. (G. Koehler et al., Nature, 256, 495-497, published in 1975) or a tumorigenic cell by a virus such as Epstan-Barr virus. Can be obtained.
[0060]
Preparation of a monoclonal antibody by the cell fusion method can be performed by the following operation.
First, a mammal (mouse, nude mouse, rat, etc., for example, BALB / c of an inbred mouse) or a bird (chicken, etc.) is immunized with the immunogen or a conjugate of the immunogen and a carrier. . The immunization amount of the immunogen or the conjugate of the immunogen and the carrier can be appropriately determined depending on the type of immunized animal, the site of immunization, and the like. Preferably, 0.1 μg to 5 mg of the immunogen or a combination of the immunogen and carrier is immunized per dose.
[0061]
The immunogen or the conjugate of the immunogen and the carrier is preferably immunized by adding an adjuvant and mixing. Known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, or pertussis adjuvant can be used as the adjuvant.
[0062]
Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
After the initial immunization, booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites such as subcutaneous, intravenous, intraperitoneal or back at intervals of 1 to 2 weeks. The number of booster injections is generally 2 to 6 times. Also in this case, the immunogen or the combined immunogen and carrier is preferably boosted by adding an adjuvant and mixing.
[0063]
After the first immunization, the antibody titer in the sera of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, the immunogen or the combined immunogen and carrier is added to physiological saline. A solution dissolved in (0.9% sodium chloride aqueous solution) is injected intravenously or intraperitoneally to obtain final immunization. Three to five days after the final immunization, cells having the ability to produce antibodies such as spleen cells, lymph node cells or peripheral lymphocytes of immunized animals are obtained.
[0064]
The cell having antibody-producing ability obtained from this immunized animal is fused with myeloma cells (myeloma cells) of mammals (mouse, nude mouse, rat, etc.). A cell line deficient in an enzyme such as guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) is preferable. TIB9), P3-X63-Ag8-U1 strain (Cancer Research Research Source Bank (JCRB) 9085), P3-NS1-1-Ag4-1 strain (JCRB 0009), P3-X63-Ag8.653 strain (JCRB 0028) Alternatively, SP2 / O-Ag-14 strain (JCRB 0029) or the like is used. be able to.
[0065]
Cell fusion can be performed using a fusion promoter such as polyethylene glycol (PEG) of various molecular weights, liposomes or Sendai virus (HVJ), or by electrofusion.
[0066]
When myeloma cells are of HGPRT-deficient strain or TK-deficient strain, fusion of cells capable of producing antibodies and myeloma cells by using a selection medium (HAT medium) containing hypoxanthine / aminopterin / thymidine Only cells (hybridomas) can be selectively cultured and propagated.
[0067]
The hybridoma culture supernatant thus obtained is subjected to immunological measurement such as ELISA or Western blot using the immunogen, the combined immunogen and carrier, or human HMG-1. By measuring by this method, a hybridoma that produces an antibody that binds to human HMG-1 or the like can be selected.
[0068]
In addition, antibodies that do not bind to human HMG-2 etc. are produced by measuring the culture supernatant of the above hybridoma using an immunoassay such as ELISA or Western blot using human HMG-2 etc. The hybridoma to be selected can be selected.
[0069]
By combining these two types of hybridoma selection methods and known cloning methods such as limiting dilution, the antibody of the present invention (monoclonal antibody), ie, human HMG-1, binds to human HMG-2. Can be obtained by isolating a production cell line of an antibody that does not bind (monoclonal antibody).
[0070]
The monoclonal antibody-producing cell line is cultured in an appropriate medium, and the antibody (monoclonal antibody) of the present invention can be obtained from the culture supernatant. As the medium, a serum-free medium or a low-concentration serum medium is used. In this case, it is preferable from the viewpoint of easy purification of the antibody, and a medium such as DMEM medium, RPMI 1640 medium, or ASF medium 103 can be used.
[0071]
In addition, a monoclonal antibody-producing cell line is injected into the abdominal cavity of a mammal that is compatible with this and previously stimulated with pristane or the like, and after a certain period of time, the antibody (monoclonal antibody) of the present invention is injected from the ascites collected in the abdominal cavity. It can also be obtained.
[0072]
The monoclonal antibody thus obtained was purified by a salting-out method using ammonium sulfate, sodium sulfate or the like, a method such as ion exchange chromatography, gel filtration or affinity chromatography, or a combination of these methods. The antibody (monoclonal antibody) of the present invention can be obtained.
[0073]
2. Method for immunological measurement of human HMG-1
(1) General
The method for immunological measurement of human HMG-1 of the present invention (hereinafter sometimes referred to as “the immunological measurement method of the present invention” or “the measurement method of the present invention”) is a method of measuring human HMG-1 contained in a sample. In an immunological measurement method in which measurement is performed using an antigen-antibody reaction, “an antibody that binds to human HMG-1 but does not bind to human HMG-2” is used.
[0074]
That is, in the immunological measurement method of human HMG-1, as an antibody that binds to human HMG-1 that is a measurement target substance, “an antibody that binds to human HMG-1 but does not bind to human HMG-2”. It is the measuring method characterized by using. Thereby, in the measuring method of the present invention, HMG-2 is not measured, and an accurate measured value of HMG-1 that does not include an error can be obtained.
[0075]
This “antibody that binds to human HMG-1 but does not bind to human HMG-2” can be used without particular limitation as long as it has such properties, and the above-described effects can be obtained. it can.
[0076]
In an immunological assay using two antibodies as antibodies that bind to human HMG-1, at least one of the two antibodies binds to “human HMG-1 but human HMG-1”. Any antibody that does not bind to 2 may be used. The other may be any antibody as long as it is an “antibody that binds to human HMG-1.” Both antibodies may also be “an antibody that binds to human HMG-1 but does not bind to human HMG-2”.
[0077]
For example, in an ELISA sandwich method, one or both of an enzyme-labeled antibody and a solid-phased antibody may be “an antibody that binds to human HMG-1 but does not bind to human HMG-2”. That's fine.
[0078]
The “antibody that binds to human HMG-1 but does not bind to human HMG-2” is not limited to one type, and a plurality of types may be used simultaneously.
The details of the “antibody that binds to human HMG-1 but does not bind to human HMG-2” are described above in “1. It binds to human HMG-1 but does not bind to human HMG-2”. As described in the section “Antibodies that do not bind”.
[0079]
(2) Immunological measurement method
The measurement method of the present invention is an immunological measurement method in which human HMG-1 contained in a sample is measured using an antigen-antibody reaction. As an antibody that binds to human HMG-1 as a measurement target substance, “human As long as the antibody that binds to HMG-1 but does not bind to human HMG-2 is used, the measurement principle is not particularly limited, and the desired effect is achieved.
[0080]
Examples of the immunological measurement method include enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay (LIA), enzyme antibody method, fluorescence Antibody method, immunochromatography method, immunoturbidimetric method, latex turbidimetric method, latex agglutination measurement method, erythrocyte agglutination method, particle agglutination method, JP-A-9-229936 and JP-A-10-132919 A specific binding substance for the described measurement target substance (test substance) is immobilized, and a carrier having a surface coated with this, and a particle on which the specific binding substance for the measurement target substance (test substance) is fixed. Measurement method to be used, or ELSA method (Enzyme-linked Ligandsorbent Assay) shown by Dahlbeack et al. (Thro . B.Haemost, 79, pp. 767-772, 1998 issue; mention may be made of WO98 / 23963) and the like.
[0081]
In the immunological measurement method described above, the measurement method of the present invention can be applied to any method such as a sandwich method, a competitive method, or a homogeneous method (homogeneous method).
In addition, the measurement in the measurement method of the present invention may be performed by a method or using an apparatus such as an analyzer.
[0082]
(3) Measurement sample
Samples in the measurement method of the present invention include blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites, amniotic fluid, and other body fluids; stool; organs such as blood vessels or liver; tissues; cells; Any biological sample that may contain HMG-1, such as an extract of stool, organ, tissue, or cell, is a target.
[0083]
(4) Immunological measurement method using labeled antibody
When the measurement method of the present invention is performed by an immunological measurement method using a labeled antibody such as an enzyme immunoassay, a fluorescence immunoassay, a radioimmunoassay, or a luminescence immunoassay, a sandwich method or a competition method, etc. However, when the sandwich method is used, one of the immobilized antibody and the labeled antibody is “an antibody that binds to human HMG-1 but does not bind to human HMG-2”. Both of the immobilized antibody and the labeled antibody may be “an antibody that binds to human HMG-1 but does not bind to human HMG-2”.
[0084]
As the solid phase carrier used in the measurement method, polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, polyacrylamide, latex, liposome, gelatin, agarose, cellulose, sepharose, glass, metal, A solid support in the form of a microcapsule, a bead, a microplate (microtiter plate), a test tube, a stick, or a test piece made of a material such as ceramics or a magnetic material can be used.
[0085]
The solid phase antibody can be prepared by adsorbing and binding an antibody such as the antibody of the present invention and a solid phase carrier by a known method such as a physical adsorption method, a chemical binding method, or a combination thereof.
[0086]
In the case of physical adsorption, according to a known method, the antibody and the solid phase carrier are mixed and brought into contact with a solution such as a buffer solution, or the antibody dissolved in the buffer solution is brought into contact with the solid phase carrier, etc. It can be carried out. When the chemical binding method is used, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983; Japan Biochemical Society In accordance with a known method described in ed. "Shinsei Kagaku Kenkyu Ken 1 Protein IV", published by Tokyo Kagaku Dojin, published in 1991, etc., the antibody and solid phase carrier are divalent crosslinking reagents such as glutaraldehyde, carbodiimide, imide ester or maleimide. It is possible to carry out the reaction by reacting with the amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the antibody and the solid phase carrier.
[0087]
Further, if it is necessary to carry out treatment to suppress non-specific reaction or spontaneous aggregation of the solid phase carrier, bovine serum albumin (BSA) is applied to the surface or inner wall surface of the solid phase carrier on which the antibody is immobilized. ), Treatment by known methods such as casein, gelatin, protein such as ovalbumin or its salt, surfactant or nonfat dry milk, etc., and blocking treatment (masking treatment) of the solid phase carrier May be.
[0088]
As the labeling substance, peroxidase (POD), alkaline phosphatase (ALP), β-galactosidase, urease, catalase, glucose oxidase, lactate dehydrogenase, amylase, or the like can be used in the case of enzyme immunoassay. In the case of fluorescence immunoassay, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate, dichlorotriazine isothiocyanate, or the like can be used. In the case of a radioimmunoassay, tritium, iodine 125, iodine 131, or the like can be used. In the luminescence immunoassay, NADH-FMNH ₂ -Luciferase system, luminol-hydrogen peroxide-POD system, acridinium ester system, dioxetane compound system, etc. can be used.
[0089]
The method for binding an antibody such as the antibody of the present invention to a labeling substance such as an enzyme is described in the Japanese Society of Clinical Pathology “Special Issue on Clinical Pathology Special Issue 53: Immunoassay for Clinical Examinations—Technology and Applications”, Clinical Pathology Publications Published in 1983; Japan Biochemical Society edited by “Neurochemistry Experiment Course 1 Protein IV”, Tokyo Kagaku Dojin, published in 1991, etc., in accordance with known methods such as glutaraldehyde, carbodiimide, imide ester or maleimide The divalent crosslinking reagent can be mixed and brought into contact with each other and reacted with the amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group or the like of each of the antibody and the labeling substance to effect the binding.
[0090]
The operation method of the measurement is a known method or the like (edited by the Japanese Society of Clinical Pathology “Special Issue on Extraordinary Clinical Pathology No. 53 Immunoassay for Clinical Examination—Technology and Applications” ”, Clinical Pathology Publications, 1983; "Enzyme Immunoassay", 3rd edition, Medical School, published in 1987; edited by Kitagawa Tsuneki et al., "Protein Nucleic Acid Enzyme Separate Volume No.31 Enzyme Immunoassay," published by Kyoritsu Publishing, 1987).
[0091]
For example, “solid phase carrier = solid phase antibody = human HMG-1 = labeled antibody” by reacting the solid phase antibody with the sample and simultaneously reacting the labeled antibody or reacting the labeled antibody after washing. To form a complex. Then, the unbound labeled antibody is washed and separated and included in the sample from the amount of labeled antibody bound to the solid phase carrier via “solid phase antibody = human HMG-1” or the amount of unbound labeled antibody. Only the amount (concentration) of HMG-1 that has been obtained can be measured.
[0092]
Specifically, in the case of an enzyme immunoassay, a substrate is reacted with an enzyme labeled with an antibody under the optimum conditions, and the amount of the enzyme reaction product is measured by an optical method or the like. In the case of the fluorescence immunoassay, the fluorescence intensity by the fluorescent substance label is measured, and in the case of the radioimmunoassay, the radiation dose by the radioactive substance label is measured. In the case of a luminescence immunoassay, the amount of luminescence by the luminescence reaction system is measured.
[0093]
(5) Immunological measurement method by agglutination method
The measurement method of the present invention can be used to produce immune complex aggregates such as immunoturbidimetry, latex turbidimetry, latex agglutination, erythrocyte agglutination, or particle agglutination, and optically transmit light and scattered light. When the measurement is carried out by a manual method or by a visual measurement method, a phosphate buffer, glycine buffer, Tris buffer, Good buffer, etc. can be used as a solvent. Reaction accelerators and nonspecific reaction inhibitors may be included.
[0094]
When an antibody such as the antibody of the present invention is used after being sensitized to a solid support, polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride are used as the solid support. -Acrylic acid ester copolymer, vinyl acetate-acrylic acid copolymer, polyacrolein, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylic acid copolymer, styrene-butadiene copolymer, methacrylic acid heavy Particles made of a material such as coalescence, acrylic acid polymer, latex, gelatin, liposome, microcapsule, erythrocyte, silica, alumina, carbon black, metal compound, metal, ceramics or magnetic material can be used.
[0095]
The method for sensitizing an antibody such as the antibody of the present invention to a solid phase carrier can be performed by a known method such as a physical adsorption method, a chemical binding method, or a combination thereof.
[0096]
In the case of physical adsorption, according to a known method, the antibody and the solid phase carrier are mixed and brought into contact in a solution such as a buffer solution, or the antibody dissolved in the buffer solution or the like is brought into contact with the solid phase carrier, etc. It can be carried out. When the chemical binding method is used, the Japanese Society of Clinical Pathology, “Special Issue on Extraordinary Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983; Japan Biochemical Society In accordance with a known method described in ed. "Shinsei Kagaku Kenkyu Ken 1 Protein IV", published by Tokyo Kagaku Dojin, published in 1991, etc., the antibody and solid phase carrier are divalent crosslinking reagents such as glutaraldehyde, carbodiimide, imide ester or maleimide. It is possible to carry out the reaction by reacting with the amino group, carboxyl group, thiol group, aldehyde group or hydroxyl group of the antibody and the solid phase carrier.
[0097]
Further, if it is necessary to carry out treatment to suppress non-specific reaction or spontaneous aggregation of the solid phase carrier, bovine serum albumin (BSA) is applied to the surface or inner wall surface of the solid phase carrier on which the antibody is immobilized. ), Treatment by known methods such as casein, gelatin, protein such as ovalbumin or its salt, surfactant or nonfat dry milk, etc., and blocking treatment (masking treatment) of the solid phase carrier May be.
[0098]
In addition, when the latex turbidimetric method is used as a measurement principle, the particle size of the latex particles used as the solid phase carrier is not particularly limited, but the latex particles are bonded and aggregated via the measurement target substance (HMG-1). For reasons such as the level of mass formation and the ease of measurement of the generated aggregate mass, the average particle size of the latex particles is preferably 0.04 to 1 μm.
[0099]
When the latex turbidimetric method is used as a measurement principle, the concentration of latex particles in which the antibody of the present invention is immobilized is included in the concentration of HMG-1 in the sample, on the latex particle surface of the antibody of the present invention. Since the optimum concentration differs depending on various conditions such as the distribution density in the sample, the particle size of the latex particles, and the mixing ratio of the sample and the measuring reagent, it cannot be generally stated.
[0100]
However, in general, a measurement reaction in which an antigen-antibody reaction is performed between “the antibody of the present invention” mixed with a sample and a latex particle and “HMG-1” contained in the sample by mixing a sample and a measurement reagent. Sometimes, the concentration of latex particles on which the antibody of the present invention is immobilized is generally 0.005 to 1% (w / v) in the reaction mixture. The concentration of “latex particles on which the antibody of the present invention is immobilized” in such a concentration as described above in the mixed solution is contained in the measurement reagent.
[0101]
In addition, when the indirect agglutination reaction method such as latex agglutination reaction method, erythrocyte agglutination reaction method or particle agglutination reaction method is used as the measurement principle, the particle size of the particles used as the solid phase carrier is not particularly limited, but the average particle The diameter is preferably in the range of 0.01 to 100 μm, and more preferably in the range of 0.5 to 10 μm. And it is preferable that the specific gravity of these particle | grains exists in the range of 1-10, and it is more preferable that it exists in the range of 1-2.
[0102]
In addition, as a container used for the measurement when an indirect agglutination reaction method such as a latex agglutination reaction method, an erythrocyte agglutination reaction method or a particle agglutination reaction method is used as a measurement principle, for example, glass, polystyrene, polyvinyl chloride, polymethacrylate, etc. And a test tube, a microplate (microtiter plate), a tray, and the like. The bottom surface of the solution storage portion (such as a well of a microplate) of these containers preferably has a shape having an inclination from the center to the periphery of the bottom, such as U-type, V-type, or UV-type.
[0103]
The measuring operation can be performed by a known method. For example, in the case of measuring by an optical method, the sample is reacted with the antibody or the antibody sensitized to the sample and the solid support, and the endpoint is measured. Transmitted light and scattered light are measured by the method or the rate method. Moreover, when measuring visually, in the said containers, such as a plate and a microplate, the antibody made to sensitize a sample and a solid-phase carrier is made to react, and the state of aggregation is visually determined. In addition, you may measure using apparatuses, such as a microplate reader, instead of measuring visually.
[0104]
3. Reagent for immunological measurement of human HMG-1
(1) General
The human HMG-1 immunological measurement reagent of the present invention (hereinafter sometimes referred to as “the immunological measurement reagent of the present invention” or “the measurement reagent of the present invention”) is the human HMG-1 contained in a sample. In the immunoassay reagent for performing measurement using antigen-antibody reaction, the antibody of the present invention described above is used, and can be used in the above-described measurement method of the present invention. Is. Accordingly, the antibody used in the measurement reagent of the present invention, the measurement principle, and the like are the same as those of the measurement method of the present invention described above.
[0105]
(2) Other reagent components
In the measurement reagent of the present invention, various aqueous solvents can be used as the solvent. Examples of the aqueous solvent include purified water, physiological saline, and various buffer solutions such as Tris buffer, phosphate buffer, or phosphate buffered saline. About the pH of this buffer solution, an appropriate pH may be appropriately selected and used. Although there is no particular limitation, it is general to select and use a pH within the range of pH 3-12.
[0106]
The measurement reagent of the present invention includes a solid phase carrier on which an antibody such as the antibody of the present invention is immobilized, a solid phase carrier sensitized with an antibody such as the antibody of the present invention, and / or the above-mentioned Proteins such as bovine serum albumin (BSA), human serum albumin (HSA), casein or a salt thereof, in addition to reagent components such as a labeled antibody obtained by binding an antibody such as the antibody of the present invention and a labeling substance such as an enzyme; Salts; Various sugars; Nonfat dry milk; Various animal sera such as normal rabbit serum; Various preservatives such as sodium azide or antibiotics; Activating substances; Reaction promoting substances; Sensitivity increasing substances such as polyethylene glycol; Nonspecific reaction suppression Substances, or one or more of various surfactants such as nonionic surfactants, amphoteric surfactants or anionic surfactants may be appropriately contained. And the density | concentration at the time of making these contain in a measuring reagent is although it does not specifically limit, 0.001 to 10% (W / V) is preferable, and 0.01 to 5% (W / V) is especially preferable. .
[0107]
Examples of the surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, Nonionic surfactants such as polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin; betaine acetate, etc. Amphoteric surfactants; or polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ether acetic acid Anionic surfactants, such as and the like.
[0108]
(3) Configuration of measurement reagent
The measuring reagent of the present invention can be used alone for measuring HMG-1 in a sample. And it can be sold by itself. Moreover, the measuring reagent of this invention can also be used for the measurement of HMG-1 in a sample in combination with another reagent. And it can also be sold in combination with other reagents. Examples of the other reagents include buffers, sample diluents, reagent diluents, reagents containing labeling substances, reagents containing substances that generate signals such as color development, and signals related to color development. A reagent containing a substance, a reagent containing a substance for performing calibration (calibration), a reagent containing a substance for performing accuracy control, and the like can be given. The other reagent is the first reagent and the measurement reagent of the present invention is the second reagent, or the measurement reagent of the present invention is the first reagent and the other reagent is the second reagent. They can be used and sold in various combinations as appropriate.
[0109]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention more specifically in detail, this invention is not limited by these Examples.
[Example 1] (Selection of amino acid sequence of human HMG-1 having high hydrophilicity and low homology with human HMG-2)
An amino acid sequence having high hydrophilicity and low homology with human HMG-2 was selected from the amino acid sequence of human HMG-1.
[0110]
(1) The amino acid sequence of human HMG-1 is the same as the data of the aforementioned Wen et al. [Wen et al., Nucleic Acids Res. , Vol. 17, pp. 1197-1214, published in 1989]
[0111]
(2) The estimation of the high hydrophilicity of each amino acid residue in the amino acid sequence of human HMG-1 was carried out by the method of Hop et al. P. Hopp et al., Proc. Natl. Acad. Sci. U. S. A. 78, 3824-3828, published in 1981]. The result is as shown in FIG.
[0112]
(3) Next, a highly hydrophilic sequence among the amino acid sequences of human HMG-1 is converted into the amino acid sequence of human HMG-2 [M. Yoshida et al. Biol. Chem. 267, 6641-6645, published in 1992]. Then, from this highly hydrophilic amino acid sequence, the amino acid sequence of human HMG-1 having low homology between human HMG-1 and human HMG-2 was selected.
[0113]
(4) Here, the first amino acid sequence selected by the present inventors is “Gly Lys” from the N-terminal amino acid residue (glycine) to the 11th amino acid residue (lysine) of human HMG-1. Gly Asp Pro Lys Lys Pro Arg Gly Lys ”. The amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” of human HMG-1 is equivalent to the amino acid sequence “Gly Lys Gly Asp Pro Asn Lys Pro Arg Gly Lys” of human HMG-2. , The sixth amino acid residue from the N-terminus is different. [In human HMG-1 it is “Lys”, whereas in human HMG-2 it is “Asn”. ]
[0114]
(5) Next, the second amino acid sequence selected by the present inventors is from the 87th amino acid residue (lysine) to the 100th amino acid residue (alanine) from the N-terminus of human HMG-1. “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala”. The amino acid sequence “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” of human HMG-1 corresponds to the amino acid sequence “Lys Lys Lys Asp Pro Asn Ala Pro Lys Arg” of human HMG-2. The 88th amino acid residue from the N-terminal is different from “Pro Pro Ser Ala”. [In human HMG-1 it is “Phe”, whereas in human HMG-2 it is “Lys”. ]
[0115]
[Example 2] (Synthesis of peptide)
The amino acid sequences “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” and “Cys Lys Phe” in which cysteine is bound to the carrier at the N-terminus of each of the two amino acid sequences selected in Example 1. The peptides “Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” were synthesized respectively.
[0116]
First, the amino acid sequence “Cys Gly Lys Gly Asp Pro Lys Lys” was applied by a t-butoxycarbonyl amino acid solid phase method according to the instruction manual using an applied biosystems model 430A peptide automatic synthesizer (Model 430A peptide synthesizer). The peptide “Pro Arg Gly Lys” was synthesized.
[0117]
In order to suppress side reactions, the peptide synthesized from the resin was eliminated by the hydrogen fluoride method in the presence of dimethylsulfide, p-thiocresol, m-cresol and anisole as a scavenger.
Thereafter, the scavenger was extracted with dimethyl ether, and the synthesized peptide was extracted with 2N acetic acid.
[0118]
An anion exchange resin, Dowex 1-X2 (DOWEX 1-X2) was purified by anion exchange column chromatography, and the main peak was analyzed by high performance liquid chromatography (HPLC) on an octadecyl (ODS) column. The pattern was confirmed.
[0119]
And it concentrated by lyophilizing | drying with an evaporator, Then, it refine | purified by HPLC and fractionated. In addition, the apparatus and conditions at the time of this HPLC refinement | purification used the reverse phase ODS column YMC-D-ODS-5 (20 mm x 300 mm) of Yamamura Chemical Laboratory, the TWINCLE pump of JASCO Corporation, and JASCO Corporation. A gradient of 0% to 70% of acetonitrile in 0.1% trifluoroacetic acid (TFA) was performed at a flow rate of 7.0 mL / min with a GP-A40 type gradientator, and a UVIDEC-100V type detector (210 nm, manufactured by JASCO Corporation) , 1.28 AUFS).
[0120]
The synthetic peptide purified and fractionated here was lyophilized with an evaporator and concentrated.
The purity of the obtained synthetic peptide was analyzed by HPLC. The apparatus and conditions were a reverse phase ODS column YMC-R-ODS-5 (4.9 mm × 300 mm) manufactured by Yamamura Chemical Laboratory, Inc., a TWINCLE pump manufactured by JASCO Corporation, and a GP-A40 type gradient manufactured by JASCO Corporation. A gradient of 0% to 70% acetonitrile in 0.1% trifluoroacetic acid (TFA) was performed at a flow rate of 1.0 mL / min for 25 minutes, and a UVIDEC-100V detector (210 nm, 1 nm, manufactured by JASCO Corporation) was used. .28 AUFS). It was found that the synthetic peptide thus obtained had a purity of almost 100%.
[0121]
Further, in the same manner as described above, a peptide having an amino acid sequence “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” was synthesized. And when the purity of the obtained synthetic peptide was analyzed by HPLC in the same manner as described above, it was found that the purity was almost 100%.
[0122]
[Example 3] (Preparation of immunogen)
Ten milligrams of mussel hemocyanin (KLH) (manufactured by Calbiochem) or bovine serum albumin (BSA) (manufactured by Seikagaku Corporation) as a carrier is 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0). 150 μL of a 2.5% maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (Pierce) solution dissolved in N, N-dimethylformamide was added to this and stirred at room temperature for 30 minutes. Reacted.
[0123]
A Sephadex G-25 (Sephadex G-25) column which is a gel filtration column equilibrated with 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.0) at 4 ° C. Pharmacia-LCB Co., Ltd.] and monitored by absorbance at 280 nm to fractionate MBS-carrier binding components.
[0124]
The MBS-carrier binding component was adjusted to pH 7.0 with trisodium phosphate, and the peptide “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” synthesized in Example 2 was added thereto and mixed for reaction for 150 minutes. I let you.
[0125]
After the reaction, it was dialyzed 3 times against water and then freeze-dried to obtain an immunogen comprising a carrier bound to the peptide.
In addition, the peptide “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” synthesized in Example 2 was also operated as described above to obtain an immunogen comprising a carrier bound to the peptide. .
[0126]
[Example 4] (Preparation of monoclonal antibody)
Monoclonal antibodies were prepared using the immunogen prepared in Example 3 as follows.
[1] Immunity to animals
(1) Saline containing 100 μg / mL of the immunogen obtained in Example 3 (the peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” combined with KLH) (0.9% sodium chloride aqueous solution), and this was mixed with Freund's complete adjuvant in an equal amount to give an emulsion as an emulsion, subcutaneously in the abdomen of 8-week-old female BALB / c mice (Charles River Japan). 5 mL was immunized.
[0127]
(2) Two weeks after the first immunization, the above immunogen is dissolved in physiological saline to a concentration of 50 μg / mL, and this is mixed with an equal amount of Freund's incomplete adjuvant to give an emulsion. A booster injection was given. This booster injection was performed every 2 weeks.
[0128]
(3) The antibody titer in the serum of this mouse, which is an immunized animal, was measured every week from the sixth week after the first immunization by enzyme immunoassay (ELISA, EIA). The operation of this ELISA method is shown below.
(1) The immunogen obtained by binding BSA as a carrier to the peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” obtained in Example 3 at a physiological saline (5 μg / mL). 0.9% sodium chloride aqueous solution), 100 μL per well was added to a 96-well microplate (manufactured by NUNK), and allowed to stand at 37 ° C. for 2 hours to immobilize the immunogen. It was.
(2) The microplate was washed with a washing solution (phosphate buffered saline containing 0.05% Tween 20) (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2 After washing with .68 mM potassium chloride (pH 7.2)), 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.2) containing 1% BSA was added in an amount of 300 μL per well, and the mixture was incubated at 37 ° C. The mixture was allowed to stand for 2 hours for blocking, and then washed again with a washing solution.
{Circle around (3)} 100 μL of the serum of the mouse to be tested for antibody production was added to each well of this microplate as a sample, allowed to stand at 37 ° C. for 2 hours, and then washed with a washing solution.
(4) Also, as a control, 100 μL of HAT medium was added to each well of the microplate in (2) above, left at 37 ° C. for 2 hours, and then washed with a washing solution.
(5) Peroxidase (POD) -labeled anti-mouse IgG antibody (Amersham) diluted 2,000 times with phosphate buffered saline containing 3% BSA, and then microplates (3) and (4) The reaction was carried out by adding 100 μL per well and allowing to stand at 37 ° C. for 2 hours.
(6) After washing this with a washing solution, a peroxidase reaction solution (3 mM 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) [ABTS] containing 50 mM disodium hydrogen phosphate—24 mM 100 μL per well of 2 μL of 1.7% hydrogen peroxide added to 1 mL of citrate buffer immediately before use) was allowed to react at room temperature. After 15 minutes, 50 μL of 6N sulfuric acid was added per well to stop the reaction.
(7) The absorbance at 415 nm was measured using an EIA plate reader (Bio-Rad).
[0129]
(4) Since it was recognized that the antibody titer reached a plateau after 18 weeks from the first immunization, it was obtained in Example 3 in which 800 μg / mL with physiological saline was subcutaneously subcutaneously in the abdomen of this immunized mouse. 0.5 mL of the immunogen (the peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” combined with KLH) was injected. On the third day, the spleen was obtained from the mouse of this immunized animal.
[0130]
The above operations (1) to (4) are carried out by applying KLH to the peptide represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” obtained in the other Example 3. The spleen was obtained from the mouse of the immunized animal.
[0131]
[2] Myeloma cell proliferation
BALB / c mouse-derived hypoxanthine, guanine, phosphoribosyl transferase-deficient myeloma cell line P3-X63-Ag8-U1 (Cancer Research Research Source Bank 9085) containing 10% fetal bovine serum and glutamine, Proliferation was performed in RPMI 1640 tissue culture medium (Biocell) supplemented with penicillin and streptomycin.
[0132]
In this method, the myeloma cells were grown in a medium-sized bottle for cell culture (manufactured by NUNK, 200 mL) until about 80% of the bottom surface of the bottle occupied by the cells. The number of cells was counted by trypan blue dye exclusion method and hemocytometer.
[0133]
[3] Cell fusion
(1) The spleen obtained from the mouse of the immunized animal in [1] was loosened sufficiently using a stainless steel mesh # 200, and filtered while washing with RPMI1640 medium solution containing no serum. Thereafter, centrifugation was performed at 200 g to separate spleen cells. Furthermore, the spleen cells were washed again three times with RPMI 1640 medium solution without serum.
[0134]
(2) The spleen cells and the proliferated P3-X63-Ag8-U1 strain myeloma cells were mixed at a ratio of 5 to 1, followed by centrifugation. The mixed cells were slowly suspended in a RPMI 1640 medium containing 50% polyethylene glycol 1500 (PEG 1500, manufactured by Roche Diagnostics). Then, this was gradually diluted with RPMI1640 medium solution so that the polyethylene glycol concentration finally became 5%.
[0135]
(3) From this, the cells were separated by centrifugation and gradually dispersed in a growth medium consisting of S-clone medium (Sanko Junyaku Co., Ltd.) containing 5% hybridoma cloning factor (Origen). Then, in a well of a flat bottom 96-well microplate (manufactured by NUNK), 10 per well ⁶ Cells / 100 μL of cells were seeded and cultured at 37 ° C. in 5% carbon dioxide.
[0136]
(4) On the first day after cell fusion, each well was supplemented with 100 μL of HAT medium (the above growth medium supplemented with 0.01 mM hypoxanthine, 1.6 μM thymidine and 0.04 μM aminopterin, All were made by Tokyo Chemical Industry Co., Ltd.). For the next 3 days, about half of the HAT medium was replaced with fresh HAT medium every day, and thereafter, the same replacement was performed every 2 to 3 days.
[0137]
(5) The cells were observed with a microscope. Hybridoma (fusion cell) clones appear from day 10 onwards, and after 14 days, in order to examine the production of antibodies that recognize the amino acid sequence indicated by “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys”, The supernatant was screened by ELISA. The operation of this ELISA method was carried out in the same manner as in the above [1] (3).
[0138]
(6) In the screening of (5) above, a hybridoma of a well that has been found to produce an antibody that recognizes the amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” has 24 wells. The cells were spread on a plate and cultured. As the cell density increased, the small and medium-sized bottles were scaled up and cultured.
[0139]
(7) The hybridoma was cultured and maintained in an HT medium (HAT medium not containing aminopterin and hybridoma cloning factor).
[0140]
(8) When the production of an antibody recognizing the amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” was examined in the same manner as in the above (5) by ELISA, the amino acid sequence “Gly Lys Gly Asp Pro Lys Lys” was determined. It binds to the immunogen (in which the carrier is BSA) obtained in Example 3 consisting of a peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” including “Pro Arg Gly Lys”, and Identified three hybridomas producing antibodies that did not bind.
[0141]
The above operations (1) to (8) are carried out by applying KLH to the peptide represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” obtained in the other Example 3. The above-mentioned mouse spleen obtained by immunization with the above-mentioned mouse was similarly performed, and “Cys Lys Phe Lys Asp Pro Asn Ala” containing the amino acid sequence “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala”. Six hybridomas producing an antibody that binds to the immunogen obtained in Example 3 consisting of a peptide represented by “Pro Lys Arg Pro Pro Ser Ala” (carrier is BSA) and does not bind to BSA were confirmed. .
[0142]
[4] Hybridoma subcloning
(A) When the immunogen is a conjugate of a peptide and a carrier represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys”
(1) Binds to the immunogen obtained in Example 3 and comprising the peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” obtained in [3] (the carrier is BSA). Each of the hybridomas producing an antibody that does not bind to BSA was subcloned by the limiting dilution method. The number of cells of these hybridomas was counted by trypan blue dye exclusion method and hemocytometer.
These hybridomas are suspended at a ratio of 0.5 growth cells and 1 growth cell per 100 μL of HT medium. One well of a 96-well flat-bottom microplate is suspended. 100 μL was dispensed per unit. The medium was changed every 2 to 3 days to grow the hybridoma.
[0143]
(2) Two weeks later, the number of colonies in each well was examined under a microscope, and the immunogen obtained in Example 3 consisting of a peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” ( The hybridoma producing an antibody that binds to the carrier with BSA and does not bind to BSA was examined by ELISA in the same manner as described above. One colony was present in one well, and two hybridomas (wells) producing such an antibody could be obtained.
[0144]
(3) This was transferred to a 24-well plate and cultured for 2 weeks until cell growth was good.
[0145]
(4) Next, the reactivity of the antibodies produced by these hybridomas with human HMG-1 prepared in Reference Example 2 was examined by ELISA. In this ELISA method, the solid phase in a 96-well microplate is replaced with the human HMG-1 prepared in Reference Example 2, and the sample is replaced with the culture supernatant of each hybridoma (each well). Except for this, the procedure was the same as in (3) of [1] above.
As a result, it was found that one of the hybridomas is a cell line that produces an antibody that binds to the human HMG-1.
[0146]
(5) This hybridoma was cloned again in the same manner as in the above (1) and (2), and antibody production was examined for each well. One colony hybridoma was present in each well, and The immunogen obtained in Example 3 consisting of a peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” containing the amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” One hybridoma producing an antibody that binds to BSA) and does not bind to BSA was obtained.
[0147]
(6) The reactivity of the antibody produced by this hybridoma clone with the human HMG-1 was examined again by ELISA as in (4) above. As a result of this study, it was confirmed that the antibody (monoclonal antibody) produced by this hybridoma clone was an antibody (monoclonal antibody) that binds to human HMG-1.
[0148]
(7) Next, the reactivity of the antibody produced by this hybridoma with each of bovine HMG-1 and bovine HMG-2 prepared in Reference Example 3 was examined by ELISA. In this ELISA method, the solid phase in a 96-well microplate was changed to bovine HMG-1 or bovine HMG-2 prepared in Reference Example 3, and the sample was mixed with this hybridoma (this well). The procedure was the same as in the above [1] (3) except that the culture supernatant was replaced.
[0149]
As a result of this examination, it was confirmed that the antibody produced by this hybridoma binds to bovine HMG-1, but does not bind to bovine HMG-2.
The amino acid sequence “Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” adopted as the amino acid sequence of the immunogen peptide and the amino acid sequence “Gly Lys Gly Asp Pro” of HMG-2 corresponding to this sequence Asn Lys Pro Arg Gly Lys is the same sequence in both human and bovine.
[0150]
Therefore, from the above, it was revealed that the antibody (monoclonal antibody) produced by this hybridoma is an antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2. .
[0151]
This hybridoma was named MD77 and was registered as FERM P-18404 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1-6 East 1st, Tsukuba, Ibaraki, Japan) July 4, 2001 Deposited at the end.
[0152]
(B) When the immunogen is a conjugate of a peptide and a carrier represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala”
(1) The immunogen obtained in Example 3 consisting of the peptide represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” obtained in [3] above (the carrier is BSA) Each of the hybridomas producing an antibody that binds to (B) and does not bind to BSA was subcloned by the limiting dilution method. The number of cells of these hybridomas was counted by trypan blue dye exclusion method and hemocytometer.
[0153]
These hybridomas are suspended at a ratio of 0.5 growth cells and 1 growth cell per 100 μL of HT medium. One well of a 96-well flat-bottom microplate is suspended. 100 μL was dispensed per unit. The medium was changed every 2 to 3 days to grow the hybridoma.
[0154]
(2) Two weeks later, the number of colonies in each well was examined under a microscope, and obtained in Example 3 consisting of a peptide represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” Hybridomas that produce antibodies that bind to the immunogen (carrier BSA) but not to BSA were examined by ELISA as described above. One colony was present in one well, and four hybridomas (wells) producing such an antibody could be obtained.
[0155]
(3) This was transferred to a 24-well plate and cultured for 2 weeks until cell growth was good.
[0156]
(4) Next, the reactivity of the antibodies produced by these hybridomas with human HMG-1 prepared in Reference Example 2 was examined by ELISA. In this ELISA method, the solid phase in a 96-well microplate is replaced with the human HMG-1 prepared in Reference Example 2, and the sample is replaced with the culture supernatant of each hybridoma (each well). Except for this, the procedure was the same as in (3) of [1] above.
As a result, it was found that one of the hybridomas is a cell line that produces an antibody that binds to the human HMG-1.
[0157]
(5) This hybridoma was cloned again in the same manner as in the above (1) and (2), and antibody production was examined for each well. One colony hybridoma was present in each well, and Obtained in Example 3 consisting of a peptide represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” containing the amino acid sequence “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” Two hybridomas were produced that produced antibodies that bound to the immunogen (carrier BSA) and did not bind to BSA.
[0158]
(6) The reactivity of each antibody produced by these two hybridoma clones with human HMG-1 was examined again by ELISA as in (4). As a result of this examination, it was confirmed that each antibody (monoclonal antibody) produced by these two hybridoma clones was an antibody (monoclonal antibody) that binds to the human HMG-1.
[0159]
(7) Next, the reactivity of each antibody produced by these two hybridomas with each of bovine HMG-1 and bovine HMG-2 prepared in Reference Example 3 was examined by ELISA. In this ELISA method, the solid phase in a 96-well microplate was changed to bovine HMG-1 or bovine HMG-2 prepared in Reference Example 3, and the samples were hybridomas (this well). The procedure was the same as in (3) of [1] except that the culture supernatant was replaced.
As a result of this examination, it was confirmed that each antibody produced by these two hybridomas binds to both bovine HMG-1 and bovine HMG-2.
[0160]
The amino acid sequence “Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” adopted as the amino acid sequence of the immunogen peptide, and the amino acid sequence “Lys Lys” of HMG-2 corresponding to this sequence “Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” is the same sequence in both human and bovine.
[0161]
Therefore, from the above, it is clear that each antibody (monoclonal antibody) produced by these two hybridomas is an antibody (monoclonal antibody) that binds to both human HMG-1 and human HMG-2. became.
[0162]
These hybridomas are named MD78 and are registered as FERM P-18405 at the Patent Organism Depository Center (National Institute of Advanced Industrial Science and Technology) (1st, 1st East, 1st Street, Tsukuba, Ibaraki, Japan). Deposited as of April 4th.
[0163]
[5] Production of monoclonal antibodies
(1) Each of the monoclonal antibody-producing cell lines (hybridomas) obtained in [4] above is put into a medium-sized bottle (manufactured by NUNK) one by one, and HT medium is used until about 80% of the bottom surface is occupied by cells. Culture was performed in.
(2) Thereafter, these hybridomas were scraped off and collected by centrifugation at 200 g for 5 minutes. Next, this was washed 3 times with RPMI 1640 medium containing no serum and then suspended in 2 mL of RPMI 1640 medium.
(3) Into the abdominal cavity of a male BALB / c mouse (Charles River Japan) previously treated with 2,6,10,14-tetramethylpentadecane, 1 mL of the hybridoma suspension obtained in (2) above Was injected. This was repeated again if no abdominal swelling was observed within 2 weeks of injection.
(4) Ascites was collected when swelling of the abdomen of this mouse was observed. This was centrifuged at 200 g for 5 minutes, and a supernatant containing the monoclonal antibody produced from the hybridoma was separated from the hybridoma and obtained.
[0164]
[6] Purification of monoclonal antibody
(1) To 10 mL of each supernatant liquid containing monoclonal antibody produced from hybridoma obtained in [5] above, 1.8 g of sodium sulfate was added at 22 ° C. with stirring, and the sodium sulfate was completely dissolved. Further, stirring was continued for 1 hour for salting out.
(2) This was centrifuged at 7000C (7000 g, 15 minutes), and the precipitate obtained by separating from the supernatant was dissolved in 2 mL of 40 mM sodium phosphate buffer (pH 8.0) containing 30 mM sodium chloride. .
(3) Next, this was sufficiently dialyzed against 40 mM sodium phosphate buffer (pH 8.0) containing 30 mM sodium chloride, and then centrifuged at 1000 g for 20 minutes to remove insoluble matters.
(4) A flow rate of 0.4 mL / min was applied to a DEAE-cellulose ion exchange column (manufactured by Selva) [1 × 10 cm] which had been equilibrated with a 40 mM sodium phosphate buffer (pH 8.0) containing 30 mM sodium chloride. And 2 mL of the eluate was collected.
(5) It was confirmed from the absorbance at 280 nm that immunoglobulin G (IgG) was contained in the flow-through fraction of the eluate, and this was collected and concentrated to 2 mL.
(6) Furthermore, this was subjected to protein A-Sepharose CL-4B affinity chromatography (Pharmacia-LCB Co., Ltd.) for purification to obtain a purified monoclonal antibody.
[0165]
In the case where the immunogen is a conjugate of the peptide represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys” and a carrier, it binds to human HMG-1, but binds to human HMG-2. The amount of antibody not obtained (monoclonal antibody) was 0.5 mg as the amount of protein.
[0166]
In addition, it binds to both human HMG-1 and human HMG-2 when the immunogen is a conjugate of a peptide and a carrier represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” The obtained amount of antibody (monoclonal antibody) was 0.4 mg as the amount of protein.
[0167]
[Example 5] (Preparation of peroxidase-labeled antibody)
An antibody that binds to human HMG-1 obtained in Example 4 but does not bind to human HMG-2 was labeled with peroxidase to prepare a peroxidase-labeled antibody.
(1) Introduction of maleimide group into peroxidase
1 mg of N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid was dissolved in 0.3 mL of 0.1 M phosphate buffer (pH 7.0) after dissolving 4 mg of peroxidase (derived from horseradish). A solution prepared by dissolving 0 mg in 60 μL of N, N′-dimethylformamide was added and reacted at 30 ° C. for 60 minutes. Thereafter, dialysis was performed overnight with 0.1 M phosphate buffer (pH 6.0). Through the above operation, a maleimide group was introduced into the peroxidase.
[0168]
(2) Introduction of thiol group into antibody
In the case where the immunogen obtained in Example 4 is a conjugate of a peptide and a carrier represented by “Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys”, it binds to human HMG-1, but it binds to human HMG-1. -2 to 0.5 mL of a 0.1 M phosphate buffer solution (pH 6.5) containing an antibody that does not bind to -2 (monoclonal antibody) at a concentration of 10 mg / mL. 6 mg of N, N′-dimethylformamide dissolved in 10 μL was added and reacted at room temperature for 30 minutes.
[0169]
Thereafter, 20 μL of 0.1 M EDTA, 0.1 mL of 0.1 M Tris-HCl buffer (pH 7.0), and 0.1 mL of 1 M hydroxylamine hydrochloride (pH 7.0) were added thereto. And left at 30 ° C. for 5 minutes.
[0170]
This was then passed through a Sephadex G-25 column that had been equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA and subjected to simple gel filtration chromatography. The acetylmercaptosuccinic anhydride was removed and the antibody fraction was collected.
Through the above operation, a thiol group was introduced into an antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2.
[0171]
(3) Preparation of labeled antibody
The peroxidase introduced with the maleimide group prepared in the above (1) and the antibody introduced with the thiol group prepared in the above (2) were mixed one-on-one and reacted at 30 ° C. for 20 hours to produce peroxidase to the antibody. Was introduced (labeled). This was then passed through a column of Ultragel AcA34 that had been equilibrated with 0.1 M phosphate buffer (pH 6.5) and subjected to gel filtration chromatography. Each fraction of the gel filtration chromatography was confirmed by 10% polyacrylamide electrophoresis, and only the fraction of the antibody bound with peroxidase was collected so that unbound peroxidase was not mixed. The antibody fraction bound to peroxidase was concentrated to obtain a peroxidase-bound antibody, that is, a peroxidase-labeled antibody. And the protein concentration of the solution containing this peroxidase labeled antibody was measured.
[0172]
[Example 6] (Microplate immobilized antibody)
The antibody that binds to both human HMG-1 and human HMG-2 obtained in Example 4 was immobilized on a microplate to prepare a microplate-immobilized antibody.
(1) Human HMG-1 and human when the immunogen obtained in Example 4 is a conjugate of a peptide and a carrier represented by “Cys Lys Phe Lys Asp Pro Asn Ala Pro Lys Arg Pro Pro Ser Ala” Antibodies that bind to both HMG-2 (monoclonal antibodies) were added to phosphate buffered saline (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2.68 mM potassium chloride ( After adjusting the pH to 15 μg / mL according to pH 7.2)), add 100 μL per well to a 96-well microplate (manufactured by NUNK), and let stand at 37 ° C. for 2 hours. And adsorbed to solid phase.
(2) This antibody-immobilized microplate was washed with a washing solution (phosphate buffered saline (pH 7.2) containing 0.05% Tween 20) and then 10 mM containing 1% BSA. Potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.2) was added in an amount of 300 μL per well, allowed to stand at 37 ° C. for 2 hours for blocking, and then washed again with a washing solution.
[0173]
Through the above operation, a microplate-immobilized antibody was prepared by immobilizing an antibody (monoclonal antibody) that binds to both human HMG-1 and human HMG-2 on a microplate.
[0174]
[Example 7] (Immunoassay reagent and immunoassay method for human HMG-1 using an antibody that binds to human HMG-1 but does not bind to human HMG-2)
The peroxidase-labeled antibody prepared in Example 5 and the microplate immobilized antibody prepared in Example 6 were used as immunological measurement reagents, and were prepared in human HMG-1 prepared in Reference Example 2 and Reference Example 3. Measurement was performed by enzyme immunoassay (sandwich method) of bovine HMG-1. Then, a calibration curve in this immunological measurement method was prepared.
[0175]
1. Reagent
(1) Peroxidase-labeled antibody
A peroxidase-labeled antibody prepared by linking the peroxidase to an antibody that binds to human HMG-1 but does not bind to human HMG-2, prepared in Example 5, was used as an enzyme-labeled antibody in the sandwich method of enzyme immunoassay. Used as.
(2) Microplate immobilized antibody
The microplate-immobilized antibody prepared in Example 6 in which the antibody that binds to both human HMG-1 and human HMG-2 was immobilized on each well of the microplate was immobilized on the sandwich method of the enzyme immunoassay method. Used as phased antibody.
(3) Cleaning fluid
Phosphate buffered saline (pH 7.2) containing 0.05% Tween 20 (Tween 20) was prepared and used as a washing solution.
(4) Peroxidase substrate solution
Immediately before use, 2 μL of 1.7% hydrogen peroxide for 1 mL of 50 mM disodium hydrogen phosphate-24 mM citrate buffer containing 3 mM 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) The added one was prepared and used as a peroxidase substrate as a label, that is, a peroxidase substrate solution.
(5) Reaction stop solution
A 6N aqueous sulfuric acid solution was prepared and used as a reaction stop solution.
[0176]
2. sample
(1) Sample containing human HMG-1
The solution containing human HMG-1 prepared in Reference Example 2 was sufficiently dialyzed against 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide. The protein concentration of the solution containing the human HMG-1 after dialysis was determined by a protein assay (manufactured by Bio-Rad). Then, the solution containing the human HMG-1 is diluted with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide, and the human HMG- Samples having a concentration of 360 ng / mL, 720 ng / mL, or 1,080 ng / mL were prepared, respectively.
(2) Sample containing bovine HMG-1
The solution containing bovine HMG-1 prepared in Reference Example 3 was sufficiently dialyzed against 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide. The protein concentration of the solution containing bovine HMG-1 after dialysis was determined by a protein assay (Bio-Rad). Then, the solution containing bovine HMG-1 was diluted with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide to obtain bovine HMG-1 concentration. Prepared samples of 10 ng / mL, 50 ng / mL, 100 ng / mL or 500 ng / mL, respectively.
(3) 0 ng / mL sample
A 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution (pH 7.4) containing 0.01% sodium azide was added to a sample having a human HMG-1 concentration and a bovine HMG-1 concentration of 0 ng / mL. did.
[0177]
3. Measurement by enzyme immunoassay (sandwich method)
(1) The sample containing 3 types of human HMG-1 prepared in 2 above, the sample containing 4 types of bovine HMG-1 and the sample of 0 ng / mL were each diluted 2-fold with physiological saline. did.
(2) Each sample diluted in (1) above was added with 100 μL to the well of the microplate-immobilized antibody in (1) and allowed to stand at 37 ° C. for 2 hours to immobilize the antibody on the microplate. And antigen-antibody reaction with HMG-1 contained in the sample.
(3) Next, each well of the microplate-immobilized antibody was washed with the washing solution of 1.
(4) The 1 peroxidase-labeled antibody was diluted 1,000 times with phosphate buffered saline containing 3% BSA. Next, 100 μL of each was added to each well of the microplate immobilized antibody subjected to the washing operation of (3) above, and then allowed to stand at 37 ° C. for 2 hours. As a result, a reaction for binding the peroxidase-labeled antibody to HMG-1 bound to the antibody immobilized on the microplate was performed.
(5) Thereafter, each well of the microplate-immobilized antibody was washed with the washing solution of 1.
(6) Next, 100 μL of the above-mentioned peroxidase substrate solution was added to each well of the microplate-immobilized antibody. And it was made to react at room temperature.
(7) 15 minutes after the addition of the peroxidase substrate solution, 100 μL of the reaction stop solution of 1 was added to each well of the microplate-immobilized antibody to stop the reaction of the labeled peroxidase. .
(8) Next, the absorbance (450 nm) of the solution in each well of the microplate-immobilized antibody was measured with a microplate reader (Bio-Rad).
(9) The measured values, that is, calibration curves, of the samples obtained by the above operation are shown in the figure. The measured values of the sample containing human HMG-1 and the sample of 0 ng / mL, that is, a calibration curve are shown in FIG. Moreover, the measured value of the sample containing the bovine HMG-1 and the sample of 0 ng / mL, that is, a calibration curve is shown in FIG.
[0178]
In these figures, the horizontal axis represents the concentration of human HMG-1 or bovine HMG-1 contained in the sample, and the vertical axis represents the measured absorbance value at 450 nm. However, the measured value of absorbance was obtained by subtracting the absorbance of phosphate buffered saline (pH 7.2) containing 3% BSA as a blinded value.
[0179]
4). Summary
From FIG. 2, in the sample containing the human HMG-1, the absorbance obtained in proportion to the contained human HMG-1 concentration is increased, and the measurement is proportional to the human HMG-1 concentration contained in the sample. It turns out that the value can be obtained.
Therefore, it was confirmed that the human HMG-1 contained in the sample can be accurately measured by the measurement reagent and the measurement method of the present invention.
[0180]
From FIG. 3, in the sample containing the bovine HMG-1, the absorbance obtained in proportion to the contained bovine HMG-1 concentration increased, and the measurement was proportional to the bovine HMG-1 concentration contained in the sample. It turns out that the value can be obtained.
[0181]
[Example 8] (Measurement of serum sample by immunological measurement reagent and immunological measurement method of human HMG-1 of the present invention)
Regarding the immunological measurement reagent and immunological measurement method of human HMG-1 of the present invention, HMG-1 contained in a serum sample was measured to confirm the accuracy at the time of measurement of the serum sample.
[0182]
1. Reagent
“7. Peroxidase-labeled antibody”, “2) Microplate immobilized antibody”, “3) Washing solution”, “4) Peroxidase substrate solution” in “1. “(5) Reaction stop solution” was used.
[0183]
2. sample
(1) The solution containing bovine HMG-1 prepared in Reference Example 3 was sufficiently dialyzed with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide. did. The protein concentration of the solution containing bovine HMG-1 after dialysis was determined by a protein assay (Bio-Rad).
[0184]
Then, the solution containing bovine HMG-1 was diluted with 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer (pH 7.4) containing 0.01% sodium azide to obtain bovine HMG-1 concentration. Prepared solutions of 40 ng / mL, 200 ng / mL or 400 ng / mL, respectively.
[0185]
(2) The three types of solutions prepared in (1) were each diluted twice with physiological saline. And the sample which does not contain the serum whose bovine HMG-1 density | concentration is 20 ng / mL, 100 ng / mL, or 200 ng / mL, respectively was prepared.
(3) The three types of solutions prepared in (1) were each diluted 2-fold with human serum. And the serum sample whose bovine HMG-1 density | concentration is 20 ng / mL, 100 ng / mL, or 200 ng / mL, respectively was prepared.
[0186]
3. Measurement by enzyme immunoassay (sandwich method)
(1) Add 100 μL of each sample diluted in (2) to the well of the microplate-immobilized antibody in (1) above, leave it at 37 ° C. for 2 hours, and immobilize the antibody and sample on the microplate. The antigen-antibody reaction with HMG-1 contained in was carried out.
(2) Next, each well of the microplate-immobilized antibody was washed with the washing solution of 1.
(3) The 1 peroxidase-labeled antibody was diluted 1,000-fold with phosphate buffered saline containing 3% BSA. Next, 100 μL of each was added to each well of the microplate immobilized antibody subjected to the washing operation of (2) above, and then allowed to stand at 37 ° C. for 2 hours. As a result, a reaction for binding the peroxidase-labeled antibody to HMG-1 bound to the antibody immobilized on the microplate was performed.
(4) Thereafter, each well of the microplate-immobilized antibody was washed with the washing solution of 1.
(5) Next, 100 μL of the peroxidase substrate solution of 1 was added to each well of the microplate immobilized antibody. And it was made to react at room temperature.
{Circle around (6)} 15 minutes after the addition of the peroxidase substrate solution, 100 μL of the reaction stop solution of 1 was added to each well of the microplate-immobilized antibody to stop the reaction of the labeled peroxidase.
(7) Next, the absorbance (450 nm) of the solution in each well of the microplate-immobilized antibody was measured with a microplate reader (Bio-Rad).
(8) The measured values of each sample obtained by the above operation are shown in the table. The measured value of absorbance was obtained by subtracting the absorbance of phosphate buffered saline (pH 7.2) containing 3% BSA as a blinded value.
[0187]
[Table 1]

[0188]
4). Summary
From the above table, at each bovine HMG-1 concentration, the measured value (absorbance) of the serum-free sample diluted with physiological saline and the measured value (absorbance) of the serum sample diluted with human serum are almost the same. It turns out that it is.
[0189]
Serum has various components including various components, but the immunoassay reagent and immunoassay method of the present invention can control the concentration of HMG-1 even in a sample containing such serum or serum. It was confirmed that it can be measured accurately.
[0190]
As described above, the antibodies used in the immunological measurement reagents and immunological measurement methods of the present invention that were measured as described above were immunized at the same amino acid sequence between human and bovine. The immunological measurement reagent and immunological measurement method of the present invention in Example 7 can accurately measure both the human HMG-1 and the bovine HMG-1. As it has been confirmed, it is clear that the same result can be obtained even if a sample obtained by diluting human HMG-1 with serum instead of bovine HMG-1 is used as a sample.
[0191]
[Example 9] (Confirmation of reactivity of antibody with human HMG-1 and human HMG-2)
An antibody (monoclonal antibody) that binds to human HMG-1 prepared in Example 4 but does not bind to human HMG-2, binds to both human HMG-1 and human HMG-2 prepared in Example 4 Reaction of each of the antibody (monoclonal antibody) and the antibody (polyclonal antibody) that binds to both human HMG-1 and human HMG-2 prepared in Reference Example 4 with human HMG-1 and human HMG-2 Sex was confirmed by Western blot.
[0192]
1. Western blot
(1) Antibody that binds to human HMG-1 but does not bind to human HMG-2 (monoclonal antibody prepared in Example 4)
(1) Human serum was subjected to electrophoresis using a Titan gel lipoprotein electrophoresis kit (manufactured by Helena Laboratories). The support was an agarose gel, which was contacted with 2 μL of the human serum.
{Circle around (2)} Then, barbital buffer (pH 8.8) was used as the electrophoresis buffer, and electrophoresed by applying electricity at a voltage of 90 V for 75 minutes. The electrophoresis was performed by preparing two sets of the same ones, and the following operations were performed in the same manner.
(3) The transfer after the electrophoresis in (2) was performed by a dry method using a Nova blot electrophoretic transfer kit (Pharmacia-LCB) according to the instruction manual.
[0193]
First, the agarose gel subjected to electrophoresis in (2) was placed on a transfer device. Next, on this agarose gel, a 9 cm × 9 cm nitrocellulose membrane (manufactured by Bio-Rad) was overlaid, and 48 mM tris (hydroxymethyl) aminomethane, 39 mM glycine, 0.0375% (W / V) sodium dodecyl sulfate. Transfer was performed at a current of 65 mA for 2 hours using a transfer buffer solution (SDS) and 20% (V / V) methanol.
(4) The nitrocellulose membrane subjected to this transfer was subjected to phosphate buffered saline containing 1% BSA (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride and 2.68 mM. Blocking was performed by immersing in 20 mL of an aqueous solution containing potassium chloride (pH 7.2) at 4 ° C. overnight.
(5) Next, this was washed with shaking in 20 mL of a washing solution (phosphate buffered saline containing 0.05% Tween 20) for 10 minutes. This operation was performed three times.
(6) An antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2 prepared in Example 4 was dissolved in 20 mL of phosphate buffered saline and dissolved in this solution. The nitrocellulose membrane subjected to the above operation (5) was reacted by being immersed for 2 hours at room temperature.
(7) The nitrocellulose membrane subjected to the above operation (6) was washed with shaking in 20 mL of a washing solution for 10 minutes. This was done three times.
(8) Next, peroxidase-labeled anti-mouse IgG antibody (manufactured by Dako) was diluted 500-fold with phosphate buffered saline containing 3% BSA to prepare a 20 mL solution. The nitrocellulose membrane of No. 7 was immersed and reacted at room temperature for 2 hours.
{Circle around (9)} The nitrocellulose membrane was washed with shaking in 20 mL of a washing solution for 10 minutes. This operation was performed three times.
[10] The nitrocellulose membrane of (9) above is added to 20 mL of phosphate buffered saline containing 0.025% 3,3′-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide at room temperature. Color was developed by dipping for a minute.
[0194]
By the above operation, the results of Western blotting for the antibody (monoclonal antibody) prepared in Example 4 that binds to human HMG-1 but does not bind to human HMG-2 were obtained.
[0195]
(2) Antibody that binds to both human HMG-1 and human HMG-2 (monoclonal antibody prepared in Example 4)
“An antibody that binds to human HMG-1 but does not bind to human HMG-2 (monoclonal antibody) prepared in Example 4” in (6) of (1) above was prepared in “Example 4. , Except that the antibody is changed to an antibody that binds to both human HMG-1 and human HMG-2 (monoclonal antibody). The results of Western blotting were performed on the prepared antibody (monoclonal antibody) that binds to both human HMG-1 and human HMG-2.
[0196]
(3) Antibody that binds to both human HMG-1 and human HMG-2 (polyclonal antibody prepared in Reference Example 4)
The antibody prepared in Example 4 that binds to human HMG-1 but does not bind to human HMG-2 (monoclonal antibody) in (6) of (1) above was prepared in “Reference Example 4. , Except that it is changed to “Antibodies that bind to both human HMG-1 and human HMG-2 (polyclonal antibodies)”, the procedure of Reference Example 4 was repeated. The results of Western blotting were performed on the prepared antibody (polyclonal antibody) that binds to both human HMG-1 and human HMG-2.
[0197]
2. Experimental result
(1) Western blot results
The results of Western blotting in (1), (2) and (3) above are shown in FIG.
In this figure, “MA1” is the result for an antibody that binds to human HMG-1 but does not bind to human HMG-2 (the monoclonal antibody prepared in Example 4), and “MA2” is human HMG. 1 is a result of an antibody that binds to both human-1 and human HMG-2 (monoclonal antibody prepared in Example 4), and “PA” is an antibody that binds to both human HMG-1 and human HMG-2 (Reference Example) 4 is a result of the polyclonal antibody prepared in 4.).
[0198]
In this figure, the band “1” is a band due to human HMG-1 contained in human serum, and the band “2” is a band due to human HMG-2 contained in human serum.
[Separately, instead of human serum, purified bovine HMG-1 and bovine HMG-2 were each electrophoresed and subjected to Western blotting, and confirmed from the position of the obtained band. . (The homology of the amino acid sequences of human HMG-1 and bovine HMG-1, and human HMG-2 and bovine HMG-2 are so high that they migrate at approximately the same position in electrophoresis.)]
[0199]
(2) Control
With respect to each of the other nitrocellulose membranes which have been subjected to the operation (5) of (1) in the above (1), (2) and (3), the operation (6) of (1) is performed. However, the operation of (1) below (7) was performed in the same manner, and this was used as a control.
[0200]
In this control (control) in which neither an antibody that binds to human HMG-1 but does not bind to human HMG-2 nor an antibody that binds to both human HMG-1 and human HMG-2 is allowed to act. No color development was observed at any of the positions where the human HMG-1 band appeared and the positions where the human HMG-2 band appeared.
From this, it was confirmed that non-specific color development did not occur in each Western blotting method.
[0201]
3. Summary
From FIG. 4, it binds to both the human HMG-1 and human HMG-2 prepared in Example 4 and the antibody (monoclonal antibody) that binds to both human HMG-1 and human HMG-2 prepared in Example 4. In the antibody (polyclonal antibody), color development is seen at both the position where human HMG-1 is migrated and the position where human HMG-2 is migrated.
From this, it was confirmed that both human HMG-1 and human HMG-2 exist in human serum.
[0202]
The antibody that binds to both human HMG-1 and human HMG-2 (monoclonal antibody) and the antibody that binds to both human HMG-1 and human HMG-2 (polyclonal antibody) are certainly human HMG- It was confirmed from the results of this Western blotting that it binds to both 1 and human HMG-2.
[0203]
In addition, as shown in FIG. 4, the antibody that binds to human HMG-1 prepared in Example 4 but does not bind to human HMG-2 (monoclonal antibody) develops color at the position where human HMG-1 migrates. Although it can be seen, it can be seen that no color is seen at the position where human HMG-2 migrates.
[0204]
From this, the antibody that binds to human HMG-1 but does not bind to human HMG-2 does not bind to human HMG-2, although it does bind to human HMG-1. This was confirmed from the result of Western blotting.
[0205]
[Reference Example 1] (Preparation of human HMG-1 gene)
Human HMG-1 was cloned from human brain cDNA by DNA amplification. The PCR product was modified with BamHI and HindIII, subcloned into the BamHI-HindIII site of the sequence vector pCAL-n vector (Stratagene, California, USA), and the DNA sequence was confirmed.
[0206]
[Reference Example 2] (Preparation of human HMG-1 by DNA recombination method)
In order to express the PCR product modified with BamHI-HindIII as a fusion protein with glutathione-S-transferase, it was subcloned into the BamHI-HindIII site of the pEX expression vector.
[0207]
Next, this recombinant plasmid was transformed into E. coli. E. coli / JM1 was transformed.
After the transformed cells were cultured at about 1 L, IPTG was induced, and glutathione-S-transferase / human HMG-1 fusion protein was transformed into E. coli. It was expressed in E. coli / JM1.
[0208]
This expressed E. coli. After collecting E. coli / JM1, about 30 mL of phosphate buffered saline (PBS) [including 137 mM sodium chloride, 2.68 mM potassium chloride, 1.47 mM potassium dihydrogen phosphate and 5.59 mM disodium hydrogen phosphate Aqueous solution (pH 7.2)].
[0209]
And this was crushed by ultrasonication and the supernatant liquid was collect | recovered after centrifugation.
The glutathione-S-transferase / human HMG-1 fusion protein contained in the supernatant was purified with a glutathione column (Pharmacia).
[0210]
Next, from this purified glutathione-S-transferase / human HMG-1 fusion protein, factor Xa was allowed to act to excise human HMG-1.
Human HMG-1 was obtained and prepared by the above DNA recombination operation.
[0211]
[Reference Example 3] (Preparation of bovine HMG-1 and bovine HMG-2)
Bovine HMG-1 and bovine HMG-2 were obtained from the bovine thymus by the method of Sanders et al. Sanders et al. B. R. C. 78, 1034-1042, published in 1977].
(1) First, 500 g of bovine thymus was crushed in 600 mL of a buffer containing 140 mM sodium chloride and 0.5 mM PMSF.
(2) Next, the crushed material was centrifuged with a centrifuge, and the supernatant was removed.
{Circle around (3)} A buffer solution containing 140 mM sodium chloride and 0.5 mM PMSF was added thereto and stirred, and then centrifuged with a centrifuge to remove the supernatant. This washing operation was repeated twice.
(4) Next, 300 mL of 0.75 M perchloric acid was added to the resulting precipitate. And after centrifuging with a centrifuge, the supernatant liquid was fractionated. 400 mL of 0.75 M perchloric acid was added to the remaining precipitate. Also for this, after centrifuging with a centrifuge, the supernatant was collected. This supernatant was combined with the previously collected supernatant. The precipitate was discarded.
(5) 0.75 M perchloric acid was added to the combined supernatant to make a total volume of 1,000 mL. Next, after centrifuging with a centrifuge, the supernatant was filtered through a glass filter (grade 4).
(6) A mixed solution of 3,500 mL of acetone and 21 mL of concentrated hydrochloric acid was added to the filtrate of the above filtration. Since turbidity occurred, the mixture was centrifuged with a centrifuge and the supernatant was collected. To this supernatant, 2,500 mL of acetone was added. Then, since turbidity occurs again, this was centrifuged with a centrifuge to separate the supernatant, and the remaining precipitate was collected.
(7) The collected precipitate was naturally dried at room temperature.
[0212]
Through the above operation, approximately 200 mg of a protein fraction containing HMG-1 and HMG-2 was obtained.
(8) The protein fraction containing HMG-1 and HMG-2 is dissolved in 10 mL of 7.5 mM sodium borate buffer (pH 9.0) containing 200 mM sodium chloride, and then this 200 mM sodium chloride is contained. Dialysis was sufficiently performed with 7.5 mM sodium borate buffer (pH 9.0).
(9) After this dialysis, the product was added to a CM-Sephadex C25 column that had been equilibrated with 7.5 mM sodium borate buffer (pH 9.0).
[0213]
After that, elution was performed with 7.5 mM sodium borate buffer (pH 9.0) containing 200 mM sodium chloride, and cation exchange chromatography was performed.
This elution pattern is shown in FIG. In this figure, the vertical axis indicates the absorbance at 280 nm, and the horizontal axis indicates the number of the eluted fraction.
[0214]
[10] As a result of 20% SDS-polyacrylamide electrophoresis, the elution fraction indicated by “A” and the elution fraction indicated by “B” in FIG. It was confirmed that the elution fraction indicated by “C” and the elution fraction indicated by “D” were fractions containing bovine HMG-2.
[0215]
Accordingly, the elution fraction indicated by “A” and the elution fraction indicated by “B” in FIG. 5 are mixed and collected, and further the elution fraction indicated by “C” and the elution fraction indicated by “D”. Were collected and mixed.
[0216]
[Reference Example 4] (Preparation of polyclonal antibody)
A polyclonal antibody was prepared using bovine HMG-1 prepared in Reference Example 3 as follows.
[1] Immunity to animals
(1) The bovine HMG-1 obtained in Reference Example 3 is dissolved in physiological saline (0.9% aqueous sodium chloride solution) to 400 μg / mL, and this is mixed with Freund's complete adjuvant in equal amounts. As an emulsion, 0.5 mL of immunization was injected subcutaneously into the abdomen of a rabbit (Kitayama Labes Co., Ltd.).
[0217]
(2) Two weeks after the first immunization, the above immunogen is dissolved in physiological saline so as to be 300 μg / mL, and this is mixed with an equal amount of Freund's incomplete adjuvant to give an emulsion. A booster injection was given. This booster injection was performed every 2 weeks.
[0218]
(3) The antibody titer in the serum of this rabbit as an immunized animal was measured every week from the 6th week after the first immunization by enzyme immunoassay (ELISA, EIA). The operation of this ELISA method is shown below.
(1) Bovine HMG-1 obtained in Reference Example 3 was dissolved in physiological saline so as to have a concentration of 5 μg / mL, and this was added to a 96-well-microplate (manufactured by NUNK) at a rate of 100 μL per well at 37 ° The bovine HMG-1 was immobilized on the plate at rest for 2 hours.
(2) The microplate was washed with a washing solution (phosphate buffered saline containing 0.05% Tween 20) (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride and 2 After washing with an aqueous solution containing 68 mM potassium chloride (pH 7.2))), 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution (pH 7.2) containing 1% BSA was added in an amount of 300 μL per well. Then, the mixture was allowed to stand at 37 ° C. for 2 hours for blocking, and then washed again with a washing solution.
(3) The serum of the rabbit to be examined for antibody production is diluted 1,000-fold, 10,000-fold, and 100,000-fold with physiological saline, and 100 μL is added to each well of the microplate. The reaction was allowed to stand for 2 hours at 37 ° C., and then washed with a washing solution.
(4) As a control, 100 μL of 0.1 M phosphate buffered saline containing 1% BSA was added to each well of the microplate of (2), and allowed to stand at 37 ° C. for 2 hours. Washed.
(5) Peroxidase (POD) -labeled anti-rabbit IgG antibody (Amersham) was diluted 5,000 times with phosphate buffered saline containing 3% BSA, and then microplates (3) and (4) were used. The reaction was carried out by adding 100 μL per well and allowing to stand at 37 ° C. for 2 hours.
(6) After washing this with a washing solution, a peroxidase reaction solution (3 mM 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) [ABTS] containing 50 mM disodium hydrogen phosphate—24 mM 100 μL per well of 2 μL of 1.7% hydrogen peroxide added to 1 mL of citrate buffer immediately before use) was allowed to react at room temperature. After 15 minutes, 50 μL of 6N sulfuric acid was added per well to stop the reaction.
(7) The absorbance at 415 nm was measured using an EIA plate reader (Bio-Rad).
[0219]
(4) Since it was recognized that the antibody titer reached a plateau after 12 weeks from the first immunization, a whole blood was collected from the immunized rabbit. And antiserum was obtained from the blood of this rabbit.
(5) With respect to the antiserum obtained in the above (4), sodium sulfate was added at a ratio of 0.18 g to 1 mL of this antiserum at 22 to 25 ° C. with stirring, and after the sodium sulfate was completely dissolved Salting was performed by continuing stirring for 30 minutes.
(6) This was centrifuged (7000 g, 15 minutes) at 22 to 25 ° C., and the precipitate obtained by separating from the supernatant was dissolved in 1 mL of phosphate buffered saline.
(7) Next, this was sufficiently dialyzed against phosphate buffered saline, and then centrifuged at 1000 g for 20 minutes to remove insolubles.
(8) This was passed through a DEAE-cellulose ion exchange column (manufactured by Selva) [1 × 2.5 cm] equilibrated with phosphate buffered saline, and the eluate was collected.
(9) It was confirmed from the absorbance at 280 nm that immunoglobulin G (IgG) was contained in the flow-through fraction of the eluate, and this was collected and concentrated.
[0220]
(10) Next, this was passed through a column on which human HMG-1 prepared in Reference Example 2 was immobilized, and affinity chromatography was performed. This operation is shown below.
(1) Affinity chromatography in which 1 g of CNBr-Sepharose (manufactured by Pharmacia Biotech) was reacted with 2 mg of human HMG-1 prepared in Reference Example 2 according to the instruction manual. A chromatography column was prepared.
(2) The column was equilibrated with phosphate buffered saline, and then passed through the component (polyclonal antibody) concentrated in (9) above.
{Circle around (3)} After thoroughly washing this with phosphate buffered saline, a 0.1 M acetate buffer solution (pH 3.0) was passed therethrough.
(4) The eluted fractions were collected, dialyzed with phosphate buffered saline, and then concentrated.
[0221]
By the above affinity chromatography operation, a polyclonal antibody that binds to human HMG-1 was collected.
[0222]
(11) The rabbit polyclonal antibody obtained by the above operation is capable of binding to human HMG-1. It can also bind to human HMG-2 having high homology with human HMG-1.
[0223]
[Example 10] (Confirmation of antibody reactivity with human HMG-1 and human HMG-2)
The antibody (monoclonal antibody) that binds to human HMG-1 prepared in Example 4 but does not bind to human HMG-2 was tested for reactivity with human HMG-1 and human HMG-2 by Western blotting. Confirmed.
[0224]
In Example 9, the reactivity of human HMG-1 and human HMG-2 contained in human serum with each antibody was confirmed. In this example, human HMG prepared from human cells was used. -1 and human HMG-2 were confirmed for reactivity with the antibody.
[0225]
1. Western blot
(1) Western which is reacted with an antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2, to human HMG-1 and human HMG-2 electrophoresed and transcribed Blot method
(1) Electrophoresis of human HMG-1 and human HMG-2 prepared from human cells (HL60 cells) in Reference Example 5 described later using a Titan Gel Lipoprotein Electrophoresis Kit (manufactured by Helena Laboratories) went. The support was an agarose gel, and 7.5 mM borate buffer (pH 9) containing 200 mM sodium chloride in which human HMG-1 and human HMG-2 were dissolved to 0.5 mg / mL, respectively. 0.0) was contacted.
{Circle around (2)} Then, barbital buffer (pH 8.8) was used as the electrophoresis buffer, and electrophoresed by applying electricity at a voltage of 90 V for 75 minutes. The electrophoresis was performed by preparing two sets of the same ones, and the following operations were performed in the same manner.
(3) The transfer after the electrophoresis in (2) was performed by a dry method using a Nova blot electrophoretic transfer kit (Pharmacia-LCB) according to the instruction manual.
[0226]
First, the agarose gel subjected to electrophoresis in (2) was placed on a transfer device. Next, on this agarose gel, a 9 cm × 9 cm nitrocellulose membrane (manufactured by Bio-Rad) was overlaid, and 48 mM tris (hydroxymethyl) aminomethane, 39 mM glycine, 0.0375% (W / V) sodium dodecyl sulfate. Transfer was performed at a current of 65 mA for 2 hours using a transfer buffer solution (SDS) and 20% (V / V) methanol.
[0227]
(4) The nitrocellulose membrane subjected to this transfer was subjected to phosphate buffered saline containing 1% BSA (5.59 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodium chloride and 2.68 mM. Blocking was performed by immersing in 20 mL of an aqueous solution containing potassium chloride (pH 7.2) at 4 ° C. overnight.
(5) Next, this was washed with shaking in 20 mL of a washing solution (phosphate buffered saline containing 0.05% Tween 20) for 10 minutes. This operation was performed three times.
(6) An antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2 prepared in Example 4 was dissolved in 20 mL of phosphate buffered saline and dissolved in this solution. The nitrocellulose membrane subjected to the above operation (5) was reacted by being immersed for 2 hours at room temperature.
(7) The nitrocellulose membrane subjected to the above operation (6) was washed with shaking in 20 mL of a washing solution for 10 minutes. This was done three times.
(8) Next, peroxidase-labeled anti-mouse IgG antibody (manufactured by Dako) was diluted 500-fold with phosphate buffered saline containing 3% BSA to prepare a 20 mL solution. The nitrocellulose membrane of No. 7 was immersed and reacted at room temperature for 2 hours.
{Circle around (9)} The nitrocellulose membrane was washed with shaking in 20 mL of a washing solution for 10 minutes. This operation was performed three times.
[10] The nitrocellulose membrane of (9) above is added to 20 mL of phosphate buffered saline containing 0.025% 3,3′-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide at room temperature. Color was developed by dipping for a minute.
[0228]
By the above operation, the reactivity of the antibody (monoclonal antibody) prepared in Example 4 that binds to human HMG-1 but does not bind to human HMG-2 with human HMG-1 and human HMG-2 The result of Western blotting was obtained.
[0229]
(2) Western blotting in which human HMG-1 and human HMG-2 were electrophoresed and transcribed with an antibody (polyclonal antibody) that binds to human HMG-2
For reference, an antibody (polyclonal antibody) that binds to commercially available human HMG-2 instead of an antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2 [Product name: Anti Except that HMG-2 (C-19), Human, product number: SC-8758, manufacturer: Santa Cruz (USA), sales: Cosmo Bio Inc.) is used in the above (1) (6). ) To [10], a band was obtained and used to confirm the position of the human HMG-2 band in this Western blotting method.
[0230]
(3) Western in which bovine HMG-1 and bovine HMG-2 were electrophoresed and transcribed with an antibody (monoclonal antibody) that binds to human HMG-1 but does not bind to human HMG-2 Blot method
As a reference, in place of the above-mentioned human HMG-1 and human HMG-2, the above (1) was used except that an equal amount mixed solution of bovine HMG-1 and bovine HMG-2 prepared from bovine thymus in Reference Example 3 was used. ) To [10], a band was obtained and used for confirmation of the position of the bovine HMG-1 band in this Western blotting method.
[0231]
2. Experimental result
(1) Western blot results
The results of Western blotting in 1 above are shown in FIG.
In this figure, the lane “H (1)” is the lane of the Western blot method of (1) above, and the lane of “H (2)” is the lane of the Western blot method of (1) (2). The lane of “B (1)” is the lane of the Western blot method of (1) in (1) above.
[0232]
From FIG. 6, the following can be understood.
• Only one band is allowed in the lane “H1”.
-In the lane of “H (1)”, no band is recognized at the band position in the lane of “H (2)”.
・ The lane of “H 1” and the lane of “B 1” have a band at the same position.
[0233]
That is,
(1) It can be seen that no band is observed in the lane of “H (1)” at the position of the band of “H (2)” after reacting with an antibody that binds to human HMG-2 (polyclonal antibody). .
(2) In the lane of “H (1)” in which human HMG-1 and human HMG-2 prepared from human cells were migrated, only one band was observed, and the position of the band was human HMG- It is the same as the position of the only band in the lane of “B (1)” in which bovine HMG-1 having a high homology with 1 and bovine HMG-2 having a high homology with human HMG-2 were migrated. I understand that.
[0234]
From the above, it was confirmed that the antibody (monoclonal antibody) prepared in Example 4 binds to human HMG-1 prepared from human cells and does not bind to human HMG-2 prepared from human cells. It was.
[0235]
(2) Control
With respect to the other nitrocellulose membrane which has been subjected to the operation (1) (5) above, the operation (6) is not performed, but the operations (7) and thereafter are performed in the same manner. Was used as a control.
In (2) and (3) above, a control was prepared in the same manner.
[0236]
In the control (control) in which none of these antibodies was allowed to act, no color development was observed.
From this, it was confirmed that non-specific color development did not occur in each Western blotting method.
[0237]
[Reference Example 5] (Preparation of human HMG-1 and human HMG-2 from human cells)
Human HMG-1 and human HMG-2 were prepared from human cells (HL60 cells).
(1) First, 3 L of the supernatant of the culture solution of human cells (HL60 cells) cultured in RPMI 1640 was concentrated to about 250 mL.
(2) Next, sodium chloride was added so that the final concentration was 200 mM.
(3) This was centrifuged with a centrifuge (10,000 rpm, 30 minutes), and the supernatant was collected and filtered with a filter having a pore size of 0.45 μm.
(4) The filtrate was passed through a high trap heparin column (manufactured by Amersham Pharmacia).
(5) The column was washed with a 10 mM sodium phosphate buffer solution (pH 7.0) containing 200 mM sodium chloride.
(6) Next, a sodium phosphate buffer solution (pH 7.0) was eluted from the column with a sodium chloride concentration gradient of 200 mM to 2,000 mM.
(7) Each eluted fraction was subjected to SDS-polyacrylamide electrophoresis, and fractions containing human HMG-1 and human HMG-2 were identified based on their mobility. This fraction was a fraction eluted when the sodium chloride concentration was 500 mM to 1,000 mM.
(8) The fraction containing human HMG-1 and human HMG-2 in (7) above was applied to a CM-Sephadex C25 column equilibrated with 7.5 mM sodium borate buffer (pH 9.0). I passed.
After that, elution was performed with 7.5 mM sodium borate buffer (pH 9.0) containing 200 mM sodium chloride, and cation exchange chromatography was performed.
(9) Each fraction eluted here was subjected to SDS-polyacrylamide electrophoresis, and the fraction containing human HMG-1 and the fraction containing human HMG-2 were identified from their mobility.
By the above operation, human HMG-1 was prepared from human cells (HL60 cells), and human HMG-2 was also prepared.
[0238]
【The invention's effect】
The antibody of the present invention is an antibody that binds to human HMG-1 but does not bind to human HMG-2 that is highly homologous to human HMG-1.
[0239]
Further, the human HMG-1 immunological measurement reagent of the present invention can measure only HMG-1 without measuring HMG-2, and the measurement operation is simple and can be automated. Measurement reagent. Thereby, an accurate measurement value of HMG-1 that does not include an error can be obtained, and erroneous diagnosis can be prevented in diagnosis of diseases such as sepsis.
[0240]
And the immunological measurement method of human HMG-1 of the present invention can measure only HMG-1 without measuring HMG-2, and the measurement operation is simple and can be automated. Measurement method. Thereby, an accurate measurement value of HMG-1 that does not include an error can be obtained, and erroneous diagnosis can be prevented in diagnosis of diseases such as sepsis.
[0241]
[Sequence Listing]

[0242]
[Sequence Listing Free Text]
SEQ ID NO: 1 peptide synthesized based on the amino acid sequence of human HMG-1
SEQ ID NO: 2: Peptide synthesized based on the amino acid sequence of human HMG-1
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing the results of estimation of the hydrophilicity of each amino acid residue by the method of Hop et al.
FIG. 2 is a diagram showing a calibration curve prepared by measuring a sample containing human HMG-1 by the immunological measurement reagent and immunological measurement method of the present invention.
FIG. 3 is a diagram showing a calibration curve prepared by measuring a sample containing bovine HMG-1 by the immunological measurement reagent and immunological measurement method of the present invention.
FIG. 4 shows the results of Western blotting confirming the reactivity of each antibody with human HMG-1 and human HMG-2.
FIG. 5 shows the elution pattern of cation exchange chromatography in the preparation of bovine HMG-1 and bovine HMG-2.
FIG. 6 shows the results of Western blotting confirming the reactivity of each antibody with human HMG-1 and human HMG-2.

Claims (9)

Formula (I):
Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys (I)
Or
Formula (Ia):
Cys Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys (Ia)
An antibody that is prepared using an amino acid sequence represented by the following formula as an immunogen and binds to human high mobility group protein-1, but does not bind to human high mobility group protein-2. Claim 1, wherein the antibody is a monoclonal antibody. The antibody according to claim 2, which is a monoclonal antibody produced by the hybridoma MD77 (FERM P-18404). A peptide comprising the amino acid sequence represented by the formula (I) or (Ia) is used as an immunogen to immunize a non-human animal, and the produced antibody is obtained and bound to human high mobility group protein-1, The antibody according to claim 1 or 2, which is an antibody obtained by confirming or selecting an antibody that does not bind to high mobility group protein-2. Immunized with peptide consisting of the amino acid sequence represented in the previous above formula (I) or (Ia) to a non-human animal as an immunogen, it binds to human high mobility group protein -1, human high mobility group protein A polyclonal antibody comprising a polyclonal antibody that does not bind to -2 and a polyclonal antibody that binds to either human high mobility group protein-1 or human high mobility group protein-2, and then the resulting polyclonal The antibody is brought into contact with the solid phase carrier immobilized on the solid phase using human high mobility group protein-2 as a ligand, and binds to both human high mobility group protein-1 and human high mobility group protein-2. Polyclonal The body is bound to the solid phase via the human high mobility group protein-2 immobilized on the solid phase as a ligand to the solid phase carrier, and the solid phase carrier is passed through as a fraction without passing through the ligand. The antibody according to claim 1, which is a polyclonal antibody obtained. Immunized with peptide consisting of the amino acid sequence represented in the previous above formula (I) or (Ia) to a non-human animal as an immunogen, it binds to human high mobility group protein -1, human high mobility group protein A polyclonal antibody comprising a polyclonal antibody that does not bind to -2 and a polyclonal antibody that binds to either human high mobility group protein-1 or human high mobility group protein-2, and then the resulting polyclonal The antibody is passed through an affinity chromatography column immobilized on a solid phase with human high mobility group protein-2 as a ligand, and human high mobility group protein-1 and human high mobility group protein-2 are contacted. A polyclonal antibody that binds to both of them is bound to the solid phase via the human high mobility group protein-2 immobilized on the solid phase as a ligand to the column, and the column passes through the column without binding to the ligand. claim 1, wherein the antibody is obtained as minute polyclonal antibody. A peptide comprising the amino acid sequence represented by the formula (I) or (Ia) is used as an immunogen to immunize a non-human animal, and the produced antibody is obtained and bound to human high mobility group protein-1, The method for producing an antibody according to claim 1 or 2 , wherein the antibody is obtained by confirming or selecting an antibody that does not bind to high mobility group protein-2. Immunized with peptide consisting of the amino acid sequence represented in the previous above formula (I) or (Ia) to a non-human animal as an immunogen, it binds to human high mobility group protein -1, human high mobility group protein A polyclonal antibody comprising a polyclonal antibody that does not bind to -2 and a polyclonal antibody that binds to either human high mobility group protein-1 or human high mobility group protein-2, and then the resulting polyclonal The antibody is brought into contact with the solid phase carrier immobilized on the solid phase using human high mobility group protein-2 as a ligand, and binds to both human high mobility group protein-1 and human high mobility group protein-2. Polyclonal The body is bound to the solid phase via human high mobility group protein-2 immobilized on the solid phase as a ligand to the solid phase carrier, and the fraction passed through the solid phase carrier without binding to the ligand is obtained. The method for producing an antibody according to claim 1 , wherein the antibody is obtained. Immunized with peptide consisting of the amino acid sequence represented in the previous above formula (I) or (Ia) to a non-human animal as an immunogen, it binds to human high mobility group protein -1, human high mobility group protein A polyclonal antibody comprising a polyclonal antibody that does not bind to -2 and a polyclonal antibody that binds to either human high mobility group protein-1 or human high mobility group protein-2, and then the resulting polyclonal The antibody is passed through an affinity chromatography column immobilized on a solid phase with human high mobility group protein-2 as a ligand, and human high mobility group protein-1 and human high mobility group protein-2 are contacted. The polyclonal antibody that binds to both of them is bound to the solid phase via the human high mobility group protein-2 immobilized on the solid phase as a ligand to the column, and the column passes through the column without binding to the ligand. The method for producing an antibody according to claim 1 , wherein a fraction is obtained.

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