JP3657644B2 - Monoclonal antibodies and hybridomas - Google Patents

Description

【００５８】
〔試験例２〕
この試験は、ＳＤＳ（ドデシル硫酸ナトリウム）及び２−メルカプトエタノールにより変成させたタンパク質又は抗菌性ペプチドと抗体との交叉性を調べるために行った。
【００５９】
１）試料の調製
参考例１及び参考例２と同一の方法によりウシラクトフェリン及び抗菌性ペプチドを調製した。また、実施例１と同一の方法によりモノクローナル抗体を調製した。
【００６０】
２）試験方法
ラクトフェリン及び抗菌性ペプチドを５０ｍＭトリス−塩酸緩衝液（ｐＨ６．８）、１％ＳＤＳ（ナカライテスク社製）、０．０５％ブロモフェノールブルー（和光純薬工業社製）、５％グリセロール（ナカライテスク社製）、１％２−メルカプトエタノール（バイオラッド社製）に溶解し、１００℃で２分間加熱して変成させ、前記レムリらの方法により不連続緩衝液を用いたＳＤＳ−ポリアクリルアミドゲル（１５％）に供給し、電気泳動により分離した。
【００６１】
電気泳動ゲル上で分離したタンパク質及びペプチドをＴｒａｎｓ Ｂｌｏｔトランスファー装置（バイオラッド社製）を用い、同社の推奨する手順にしたがって、ポアサイズ０．２５μｍのニトロセルロースシート上に電気的にブロットした。ブロットしたニトロセルロースシートは１％ＢＳＡ、１０ｍＭトリス−塩酸緩衝液及び５０ｍＭ塩化ナトリウムの混合液（以下、「Ｔ10Ｎ50溶液」と記載する）で２時間緩やかに振盪しながらブロッキングした。
【００６２】
その後、上記ニトロセルロースシートを、本発明のモノクローナル抗体を含む１％ＢＳＡ、Ｔ10Ｎ50溶液中で１時間振盪し抗体を結合させた。このニトロセルロースシートを、１０ｍＭトリス−塩酸緩衝液、５０ｍＭ塩化ナトリウム及び０．０５％ノニデットＰ−４０混合液（以下、「Ｔ10Ｎ50ＮＰ40溶液」と記載する）で充分に洗浄した後、５μＣｉ ¹²⁵Ｉ標識ウサギ抗マウスＩｇＧ（アマシャム社製）及び１％ＢＳＡ及びＴ10Ｎ50溶液を含む溶液中で１時間振盪し、Ｔ10Ｎ50ＮＰ40溶液で洗浄し、乾燥させ、フィルム（コダック社製。ＸＡＲ−５）に露光させた。
【００６３】
３）試験結果
この試験の結果は、図１に示すとおりである。図１は、ニトロセルロース紙にブロットしたタンパク質のアミドブラック染色図（図１のＡ）及び本発明のモノクローナル抗体を用いた免疫染色図（図１のＢ）であり、レーン１〜３は、それぞれ分子量マーカー、ウシラクトフェリン及び抗菌性ペプチドを示す。
【００６４】
図１から明らかなとおり、本発明の抗体は抗菌性ペプチドと結合することはもちろんであるが、ＳＤＳ及び２−メルカプトエタノールで加熱変成させたラクトフェリンとも結合することが認められた。尚、モノクローナル抗体及び抗菌性ペプチドの種類を変更して試験したが、ほぼ同様の結果が得られた。
【００６５】
前記試験例１及び本試験例２から、本発明の抗体は、天然のラクトフェリンとは結合せず、抗菌性ペプチドに特異的に結合することが明らかである。
【００６６】
〔参考例１〕
イオン交換体（ＣＭ−セファロースＦＦ（ファルマシア社製））を３Ｌのカラムに充填し、４Ｌの１００ｍＭ塩酸を通液し、水洗し、イオン交換体を平衡化した。４℃に冷却したｐＨ６．９のヒト脱脂乳２２Ｌをカラムに５００ｍｌ／分の流速で通液し、透過液を回収し、再び同様にカラムに通液した。次に、５００ｍｌ／分の流速で蒸留水を通液し、１０％食塩水４Ｌを２Ｌ／分の流速で通液し、イオン交換体に吸着した塩基性タンパク質溶液３．５Ｌを得た。
【００６７】
この回収液に、硫酸アンモニウムを飽和度８０％になるように添加し、タンパク質を沈殿させ、遠心分離（３０００×ｇ）して沈殿物を回収し、飽和度８０％の硫酸アンモニウム溶液で洗浄し、脱イオン水２００ｍｌを添加して溶解し、溶解液を限外濾過膜モジュール（旭化成社製。ＳＬＰ００５３）を用いて限外濾過し、のち水を添加し、同装置を用いてダイアフィルトレーションを行い、脱塩し、凍結乾燥し、粉末状のヒトラクトフェリン約１７ｇを得た。
【００６８】
得られたヒトラクトフェリン凍結乾燥物を電気泳動法により分離し、純度を測定した結果、約９７％であった。
【００６９】
〔参考例２〕
脱脂乳から分離したウシラクトフェリン（森永乳業社製。純度約９０％）１０ｇを５％（ｗ／ｖ）の濃度で蒸留水に溶解し、１規定塩酸を添加してｐＨを３．０に調整し、ペプシン（和光純薬工業社製）を基質の３％の割合で添加し、３７℃で４時間加水分解し、のち８０℃に１５分間加熱してペプシンを失活させ、１規定水酸化ナトリウムを添加してｐＨを７．０に調整し、１５００×ｇで３０分間遠心して不溶物を除去し、凍結乾燥し、抗菌性ペプチドを含有する粉末状のペプチド混合物約７．９ｇを得た。
【００７０】
２５ｍｌのカチオン交換体（カルボキシメチルトヨパール（商標。東ソー社製。６５０Ｍ））を１００ｍＭ第一リン酸ナトリウム−第二リン酸ナトリウム緩衝液（ｐＨ７．８）で十分洗浄し、平衡化したスラリーに前記緩衝液に溶解した抗菌性ペプチド混合物溶液を添加し、ビーカー中でマグネティックスターラーで撹袢しながら十分に吸着させた。抗菌性ペプチド混合物を吸着したカチオン交換体をカラム（長さ５ｃｍ、直径３ｃｍ）に充填し、前記緩衝液を用いて、洗浄液の２８０ｎｍにおける吸光度が０．０３以下になるまで５ｍｌ／分の流速で洗浄した。同様に洗浄液の２８０ｎｍにおける吸光度が０．０３以下になるまで５ｍｌ／分の流速で１００ｍＭ塩化ナトリウム、１００ｍＭ第一リン酸ナトリウム−第二リン酸ナトリウム緩衝液（ｐＨ７．８）をカラムに通液し、非特異的にゲルに吸着したペプチドを洗浄した。
【００７１】
次に、２Ｍ酢酸アンモニウム（ｐＨ７．８）をカラムに通液し、抗菌性ペプチドを含む溶液１０ｍｌを得た。得られた抗菌性ペプチド溶液を水で平衡化した脱塩用のＰＤ１０カラム（ファルマシア社製）に５回に分けて注入し、脱塩した抗菌性ペプチド溶液を取得し、この溶液を凍結乾燥し、粉末状の抗菌性ペプチド約１８ｍｇを得た。
【００７２】
得られた精製ペプチドは、常法のアミノ酸分析、アミノ酸配列分析、元素分析及び質量分析により、配列番号１に記載するアミノ酸配列を有することを確認した。尚、配列番号１において、３番目のCys と２１番目のCys とがジスルフィド結合していると推定される。
【００７３】
〔参考例３〕
ペプチドシンセサイザー（ファルマシアLKB バイオテクノロジー社製。LKB Biolynx 4170）を使用し、シェパード等の方法［ジャーナル・オブ・ケミカル・ソサイエティー・パーキンＩ(Journal of Chemical Society Perkin Ｉ) 、第５３８頁、１９８１年］による固相ペプチド合成法に基づいて、次のようにして配列番号２に示す配列を有するペプチドを合成した。
【００７４】
NovaSynKA 誘導体樹脂（Fmoc-Cys(Acm)-NovaSynKA 。カルビオケム−ノバビオケム社製）０．１ｍｅｑ（１ｇ）を用いて、前記ペプチドシンセサイザーの合成プログラムにより脱保護基反応及び縮合反応を反復してペプチド鎖を延長した。即ち、２０％ピペリジン／ジメチルフォルムアミド（関東化学社製、以下「ＤＭＦ」と記載する）によりアミノ保護基である９−フルオレニルメトキシカルボニル（以下「Ｆｍｏｃ」と記載する）基を切断除去し、ＤＭＦで洗浄し、のちＦｍｏｃ−アミノ酸活性エステル／N-ヒドロキシベンゾトリアゾール（以下「ＨＯＢｔ」と記載する）各０．５ｍｍｏｌを反応させ、ＤＭＦで洗浄する操作を反復した。
縮合反応後、必要に応じてカイザーテストを行ないカップリングが完全であったことを確認した。合成には０．５ｍｍｏｌのカートリッジを用いた。ペプチド鎖の伸張反応が全て終了した後、２０％ピペリジン／ＤＭＦによりＦｍｏｃ基を切断し、ＤＭＦで洗浄後１０％無水酢酸／ＤＭＦでアセチル化を行なった。カイザーテストによりアセチル化が完了したことを確認した後、樹脂をＤＭＦ、tert−ペンチルアルコール（関東化学社製）、酢酸（関東化学社製）、tert−ペンチルアルコール（関東化学社製）、ＤＭＦ、ジエチルエーテル（国産化学社製）の順で十分洗浄し、真空乾燥した。
【００７５】
前記の保護ペプチド樹脂６５０ｍｇにエタンジチオール（渡辺化学社製）１．０ｍｌ、m-クレゾール（渡辺化学社製）２００ｍｌ、チオアニソール（渡辺化学社製）２．４ｍｌを室温、アルゴン気流下で１５分間攪拌し、のち氷冷下で更に１０分間攪拌した。これにトリフルオロ酢酸（渡辺化学社製、以下「ＴＦＡ」と記載する）１５ｍｌを添加して１０分間攪拌し、トリメチルシリルブロミド（渡辺化学社製）２．６ｍｌを添加して５０分間攪拌した。グラスフィルターで樹脂を濾過して除去し、濾液を直ちに減圧濃縮した。残渣に予め冷却したジエチルエーテル（国産化学社製）を添加し、遠沈管に移し、遠心分離（２５００ｒｐｍで５分間）し、上清を廃棄し、冷ジエチルエーテルを新たに添加して十分攪拌し、再び遠心分離する操作を４回反復した。ペプチド沈殿物を真空乾燥し、水に溶解して凍結乾燥を行ない、粗製ペプチド約９４．３ｍｇを得た。
【００７６】
Cys(Acm)ペプチド９４．３ｍｇにＡｇＢＦ₄ （１０ｅｑ、渡辺化学社製）／anisole （１０ｅｑ、渡辺化学社製）／ＴＦＡ溶液を加え４℃、６０分間攪拌し、ジエチルエ−テル（国産化学社製）を添加し、前記の遠心分離によるペプチドの精製を行ない、真空乾燥してＳＨ基フリ−ペプチドを得た。ＳＨ基フリ−ペプチドに５０％ＤＭＳＯ（渡辺化学社製）／１Ｎ ＨＣｌ（和光純薬社製）を加えて、室温で７時間攪拌した。ジスルフィド生成のモニタ−は高速液体クロマトグラフィー（ＨＰＬＣ）にて行った。水を添加して合成吸着剤ＨＰ２０（三菱化学社製）カラムに供給して吸着させ、水で十分洗浄し、６０％ＣＨ₃ＣＮ／１Ｎ ＡｃＯＨ（関東化学社製）でペプチドを溶出させ、遠心濃縮後水を添加して凍結乾燥し、粗製ペプチド６１．１ｍｇを得た。
【００７７】
前記粗製ペプチドを水に溶解し、遠心分離（１５０００ｒｐｍで５分間）を行ない、上清を０．４５ｍｍフィルターで濾過し、この溶液を高速液体クロマトグラフィー（ＨＰＬＣ）に供し、ペプチドを精製した。ＨＰＬＣはＬＫＢ２１５１高圧グラジエントシステム（ファルマシア社製）を用い、カラムは逆相系の市販カラム（東ソー社製。ＯＤＳ１２０Ｔ。２１．５×３００ｍｍ）を用いた。溶離液は０．１％ＴＦＡ／水をＡ液、８０％アセトニトリル／Ａ液をＢ液として、Ａ液からＢ液への濃度直線勾配により溶出した。クロマトグラムはほぼ単一のピークと認められ、相当する画分を分取した。この分取操作を数回反復し、凍結乾燥し、精製した抗菌性ペプチド約３０．５ｍｇを得た。
【００７８】
精製ペプチドは、常法のアミノ酸分析、アミノ酸配列分析、元素分析及び質量分析を実施し、目的とする配列番号２に記載のアミノ酸配列を有することを確認した。
【００７９】
〔参考例４〕
使用するアミノ酸の順序が異なることを除き、参考例３と同様の操作を行ない、配列番号３に示すアミノ酸配列を有するペプチドを合成した。アシル化保護ペプチド樹脂の収量は約５８１ｍｇ、脱保護、脱樹脂後の粗製ペプチドの収量は約８３．３ｍｇであり、精製した抗菌性ペプチド約５９．９ｍｇを得た。参考例３と同一の試験方法により配列番号３に記載のアミノ酸配列を有することを確認した。
【００８０】
〔参考例５〕
参考例１と同様に精製したヒトラクトフェリン１０ｇ（純度９７％）を用い参考例２と同様の操作を行なった。脱塩後の抗菌性ペプチドの収量は約２５ｍｇであった。
【００８１】
得られた精製ペプチドは、常法のアミノ酸分析、アミノ酸配列分析、元素分析及び質量分析により、配列番号４及び配列番号５に示すアミノ酸配列を有するジペプチドであることを確認した。尚、配列番号４に示すペプチドの９番目のCys と２６番目のCys がジスルフィド結合し、配列番号４に示すペプチドの３５番目のCys と配列番号５に示すペプチドの１０番目のCys とがジスルフィド結合していると推定される。
【００８２】
〔参考例６〕
使用するアミノ酸の順序が異なることを除き、参考例３と同様の操作を行ない、配列番号６に示すアミノ酸配列を有するペプチドを合成した。アシル化保護ペプチド樹脂の収量は約８３０ｍｇ、脱保護、脱樹脂後のCys(Acm)粗製ペプチドの収量は約２０１．０ｍｇ、酸化した粗製ペプチド１６９．２ｍｇ、精製した抗菌性ペプチド約６２．１ｍｇを得た。参考例３と同一の試験方法により配列番号６に記載のアミノ酸配列を有することを確認した。
【００８３】
〔参考例７〕
使用するアミノ酸の順序が異なることを除き、参考例３と同様の操作を行ない、配列番号７に示すアミノ酸配列を有するペプチドを合成した。アシル化保護ペプチド樹脂の収量は約５７０ｍｇ、脱保護、脱樹脂後の粗製ペプチドの収量は約５９．５ｍｇであり、精製した抗菌性ペプチド約３８．２ｍｇを得た。参考例３と同一の試験方法により配列番号７に記載のアミノ酸配列を有することを確認した。
【００８４】
〔参考例８〕
使用するアミノ酸の順序が異なることを除き、参考例３と同様の操作を行ない、配列番号８に示すアミノ酸配列を有するペプチドを合成した。アシル化保護ペプチド樹脂の収量は約７５０ｍｇ、脱保護、脱樹脂後のCys(Acm)粗製ペプチドの収量は約１８６．８ｍｇであり、酸化した粗製ペプチド１３１．２ｍｇ、精製した抗菌性ペプチド約５１．４ｍｇを得た。参考例３と同一の試験方法により配列番号８に記載のアミノ酸配列を有することを確認した。
【００８５】
〔参考例９〕
この例は、ヒトのラクトフェリン類由来ペプチドを定量するための本発明のキットに含まれるポリクローナル抗体の作成を示すものである。
【００８６】
参考例５と同一の方法により製造した配列番号４及び配列番号５に記載のアミノ酸配列を有する抗菌性ペプチドダイマー４００μｇを１．２ｍｌの生理食塩水に溶解し、１．５ｍｌのフロイント完全アジュバントと共に、４匹の１４週齢Ｊｌａ：ＪＷ系雄ウサギの皮下に１匹当たり０．５ｍｌづつ注射した。初回免疫より３週間及び５週間目に前記と同様に作成した抗菌性ペプチドダイマー溶液をフロイント不完全アジュバントと共に同じく注射した。最終免疫７日後にウサギを屠殺し血清を調製した。血清は１匹当たり１５０ｍｌ得られた。
【００８７】
ＭＡＰＳII結合緩衝液（バイオラッド社製）で平衡化したプロテインＡゲル（バイオラッド社製）２５ｍｌを充填したカラム（２０ｍｍ×８００ｍｍ）にＭＡＰＳII結合緩衝液で３倍に希釈した血清を５ｍｌ／分の流速でそれぞれアプライした。カラムはＭＡＰＳII結合緩衝液４００ｍｌで洗浄した後、ＭＡＰＳII溶出緩衝液７０ｍｌ（バイオラッド社製）で結合した抗体を溶出した。水酸化ナトリウムで直ちにｐＨを中性に調整した抗体溶液は分注し、使用するまで凍結保存した。
【００８８】
【実施例】
次に、実施例を示して本発明を更に具体的に説明するが、本発明は以下の実施例に限定されるものではない。
【００８９】
【実施例１】
この例は、合成ペプチドを抗原とした抗ウシ抗菌性ペプチドモノクローナル抗体産生ハイブリドーマの作製を示すものである。
【００９０】
(i) 抗原の作成
１６ｍｇのＫＬＨ（ピアス社製）を１ｍｌの１０ｍＭリン酸ナトリウム緩衝液（ｐＨ７．２）に溶解し、２．８ｍｇのｍ−マレイミドベンゾイル−Ｎ−ヒドロサクシニミド・エステル（m-maleimidobenzoyl-N-hydrosuccinimide ester：以下「ＭＢＳ」と記載する。和光純薬工業社製）を添加し、３０分間撹袢した。溶液を０．４５μｍのフィルターで瀘過し、セファデックスＧ−２５(Sephadex G-25：ファルマシア社製）カラムクロマトグラフィーにかけ、ＫＬＨ−ＭＢＳ溶液約５ｍｌを分取した。
【００９１】
この溶液に２．５ｍｌの０．２Ｍリン酸二ナトリウム溶液及び参考例３と同一の方法により製造した１０ｍｇの化学合成抗菌性ペプチド（配列番号２に示す配列を有する）を添加し、アルゴンガスで置換し、室温で３０分間反応させ、この溶液を免疫に使用するまで冷凍保存した。
【００９２】
(ii)マウスの感作
前記の溶液５０μｌを生理食塩水１ｍｌと混合し、１．５ｍｌのフロイント完全アジュバントと共に５匹の６週齢ＢＡＬＢ／ｃマウスの皮下に１匹当たり４００μｌを注射した。以後２週間の間隔で同様に３回免疫を行った。免疫の過程で抗原に対する血清の抗体価の上昇を、マウスから採血してＥＬＩＳＡ法により観察した。最も抗体価の高かったマウスに、１５０μｌのＰＢＳに溶解した１５μｇの配列番号２に示す化学合成抗菌性ペプチドを尾静脈から注射し、ブーストとした。
【００９３】
（iii)細胞融合
最終免疫３日後に脾細胞を無菌的に摘出し、ステンレスメッシュで単細胞にほぐし、ＲＰＭＩ１６４０（２５ｍＭ ＨＥＰＥＳ及び２ｍＭ Ｌ−グルタミン）で３回洗浄し、脾細胞の約１／４量のＸ−６３−Ａｇ８６．５．３ミエローマ細胞（ギブコ社製）と混合して遠心した。細胞のペレットに５０％ポリエチレングリコール（ＰＥＧ）１５００を添加し、細胞融合を行い、遠心した細胞ペレットに、３０％ウシ胎児血清（ギブコ社製。以下ＦＣＳと記載する）入りヒポキサンチン・チミジン（hypoxantine thymidine)培地を加え、９６穴マイクロプレート１９枚に播種し、翌日、各ウェルにＨＡＴ培地を添加した。
【００９４】
（iv) 限界希釈と抗体の特異性
融合後１３００ウェルからコロニーが生育し、これらの培養上清について、ウシラクトフェリン由来の抗菌性ペプチドを抗原とするＥＬＩＳＡ法を行い、抗原と強く反応するハイブリドーマを得た。限界希釈によるクローニングを４回反復し、安定した抗体産生を示すクローン８個を得た。
【００９５】
【実施例２】
この例は、ウシラクトフェリンから調製したウシ抗菌性ペプチドを抗原とした抗ウシ抗菌性ペプチドモノクローナル抗体産生ハイブリドーマの作製を示すものである。
【００９６】
参考例２と同一の方法により製造した配列番号１に示すウシラクトフェリン由来の抗菌性ペプチド１ｍｇを、生理食塩水１ｍｌに溶解し、１．５ｍｌのフロイント完全アジュバントと共に８匹の６週齢ＢＡＬＢ／ｃマウスの皮下に１匹当たり３００μｌの割合で注射した。以下、実施例１と同一の方法により、安定した抗体産生を示すクローン１２個を得た。
【００９７】
【実施例３】
この例は、合成ペプチドを抗原とした抗ヒト抗菌性ペプチドモノクローナル抗体産生ハイブリドーマの作製を示すものである。
【００９８】
参考例６と同一の方法により製造した配列番号６に示す抗菌性ペプチドを用いたことを除き、実施例２と同一の方法により、抗体を産生するクローン６個を得た。
【００９９】
【実施例４】
参考例４と同一の方法により製造した配列番号３に示す抗菌性ペプチドを用いたことを除き、実施例１と同一の方法により、抗体を産生するクローン８個を得た。
【０１００】
【実施例５】
参考例５と同一の方法により製造した配列番号４及び配列番号５に示す抗菌性ペプチドダイマーを用いたことを除き、実施例２と同一の方法により、抗体を産生するクローン１０個を得た。
【０１０１】
【実施例６】
参考例７と同一の方法により製造した配列番号７に示す抗菌性ペプチドを用いたことを除き、実施例１と同一の方法により、抗体を産生するクローン８個を得た。
【０１０２】
【実施例７】
参考例８と同一の方法により製造した配列番号８に示す抗菌性ペプチドを用いたことを除き、実施例２と同一の方法により、抗体を産生するクローン６個を得た。
【０１０３】
【実施例８】
この例は、ウシラクトフェリン由来の抗菌性ペプチドの定量法を示すものである。
【０１０４】
１ｍｌ当たり１ｍｇのウシラクトフェリンを含有する溶液１００ｍｌを経口摂取したヒトの胃液を５分後に回収し、回収した胃液に０．０１％の割合でペプスタチンＡ（シグマ社製）を添加し、体外でのプロテアーゼ活性によるタンパク質の分解を防止した。
【０１０５】
試験例２と同一の方法により、胃液試料及び標準の抗菌性ペプチドを電気泳動により分離し、メンブレンにブロッティングし、実施例１と同一の方法で得たクローンが産生する抗体を用いて免疫染色した。
【０１０６】
免疫染色された抗菌性ペプチドのバンドをＰＤＩ電気泳動解析システム（東洋紡社製）で定量した結果、ウシラクトフェリンを摂取したヒトの胃液中には摂取したウシラクトフェリンから生じた活性型の抗菌性ペプチドが存在すること、及びその濃度は２０μｇ／ｍｌであることが判明した。
【０１０７】
【実施例９】
この例は、抗菌性ペプチドを検出又は定量するための試薬キットを示すものである。
【０１０８】
本発明にかかるモノクローナル抗体を用い、胃液、腸内容物、便、血液、尿中のヒト抗菌性ペプチドをＥＬＩＳＡ法により検出又は定量するキットを次のとおり作製した。
【０１０９】
本発明のキットは、参考例９と同一の方法により製造した抗ヒト抗菌性ペプチドポリクローナル抗体プレコート９６穴マイクロプレート、リン酸緩衝液、実施例５と同一の方法により製造した抗ヒト抗菌性ペプチドモノクローナル抗体、洗浄液、ペルオキシダーゼ結合抗マウス抗体、ヒト抗菌性ペプチドスタンダード溶液、及び０．１％濃度のｏ−フェニレンジアミン溶液からなり、それぞれ個別に収納されている。
【０１１０】
【発明の効果】
以上詳述したとおり、本発明は、ウシ若しくはヒトのラクトフェリン類の断片、例えば抗菌性ペプチド等と結合し、天然のラクトフェリンとは結合しないモノクローナル抗体、該抗体を産生するハイブリドーマ、ウシ若しくはヒトのラクトフェリン類の断片の検出又は定量方法、及び該検出又は定量方法に使用する試薬キットに関するものであり、本発明によって奏せられる効果は、次のとおりである。
【０１１１】
１）ヒト若しくはウシ由来のラクトフェリン類の断片に対して特異性の高い抗体及びこれを産生する細胞ラインが得られる。
２）複雑な装置等を使用することなく、ヒト若しくはウシ由来のラクトフェリン類の断片、例えば胃液、腸内容物、便、血液、尿等に含有されるラクトフェリン類由来の抗菌性ペプチドを、特異的、かつ高感度に検出又は定量することができる。
【０１１２】
【配列表】

【０１１３】

【０１１４】

【０１１５】

【０１１６】

【０１１７】

【０１１８】

【０１１９】

【図面の簡単な説明】
【図１】 電気泳動後のタンパク質をブロットしたニトロセルロース紙の、アミドブラック染色図（図１のＡ）及び本発明のモノクローナル抗体を用いた免疫染色図（図１のＢ）である。[0001]
[Industrial application fields]
The present invention relates to a monoclonal antibody that specifically binds to a bovine or human lactoferrin fragment, a hybridoma that produces the monoclonal antibody, a method for detecting or quantifying the lactoferrin fragment using the monoclonal antibody, and the monoclonal antibody The present invention relates to a reagent kit for detecting or quantifying a fragment of lactoferrin to be used.
[0002]
[Prior art]
Monoclonal antibodies against natural bovine and human lactoferrin have already been disclosed (for example, Japanese Patent Publication No. 6-69370 and Japanese Patent Publication No. 6-61263), but these antibodies can be obtained using natural lactoferrin as an antigen. Monoclonal antibody that binds to lactoferrin but does not bind to antimicrobial peptides present in hydrolysates of lactoferrins. Moreover, there is no report of the antibody couple | bonded with the antimicrobial peptide obtained from the hydrolyzate of lactoferrin.
[0003]
The antibacterial peptide is an antibacterial peptide isolated from a hydrolyzate of bovine or human lactoferrin, and is already known (Japanese Patent Laid-Open No. 5-92994).
[0004]
Orally ingested lactoferrin is considered to be digested by digestive enzymes secreted into the gastric juice, such as pepsin, to produce antibacterial peptides with strong antibacterial activity. Thus, in order to detect and quantify a bioactive peptide newly produced from a protein hydrolyzed by an enzyme, an antibody obtained using the protein before hydrolysis as an antigen is insufficient. The reason is that when a monoclonal antibody is prepared using the protein before hydrolysis as an antigen, not only a bioactive peptide but also a peptide that does not have antibacterial activity or a protein that binds to a protein without antibacterial activity may be obtained. Because it is expensive.
[0005]
From such a viewpoint, an antibody capable of recognizing only an antibacterial peptide having high activity without binding to lactoferrin has been desired.
[0006]
[Problems to be solved by the invention]
The present invention has been made in view of the above prior art, and an object of the present invention is to provide a monoclonal antibody that specifically binds to a fragment of human or bovine lactoferrins.
[0007]
Another object of the present invention is to provide a hybridoma producing a monoclonal antibody that specifically binds to a fragment of human or bovine lactoferrins.
Another object of the present invention is to provide a method for detecting or quantifying fragments of human or bovine lactoferrins.
[0008]
Yet another object of the present invention is to provide a kit for detecting or quantifying fragments of human or bovine lactoferrins.
[0009]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have succeeded in producing a monoclonal antibody that specifically binds to a fragment of human or bovine lactoferrin, particularly an antimicrobial peptide, The present invention has been completed.
[0010]
  That is, the first invention of the present invention for solving the above-mentioned problem isA monoclonal antibody having a chemically synthesized antibacterial peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 as an antigen, the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2Combined withSeparated from skim milkMonoclonal antibody that does not bind to lactoferrinis there.
[0011]
  The second invention of the present invention for solving the above-mentioned problem isA monoclonal antibody having a chemically synthesized antibacterial peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 as an antigen, the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2Combined withSeparated from skim milkA hybridoma that produces monoclonal antibodies that do not bind lactoferrin.is there.
[0012]
  The third invention of the present invention for solving the above-mentioned problems isA peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 contained in a sample is immunologically measured using the monoclonal antibody described in claim 1 from the amino acid sequence shown in SEQ ID NO: 1. A method for detecting or quantifying a peptide comprising:Next a) or a);
  A)A peptide comprising the amino acid sequence set forth in SEQ ID NO: 1A diluted sample containingClaim 1Adding a secondary antibody in which an antibody against immunoglobulin of an immunized animal used for the preparation of the monoclonal antibody is labeled with a labeling substance, and detecting the labeling substance,
  B)A peptide comprising the amino acid sequence set forth in SEQ ID NO: 1A diluted sample containingClaim 1Adding a labeled monoclonal antibody obtained by labeling the monoclonal antibody described in 1 with a labeling substance, and detecting the labeling substance,A peptide comprising the amino acid sequence set forth in SEQ ID NO: 1To detect or quantifyThe lawThis is a preferred embodiment.
[0013]
  The fourth invention of the present invention for solving the above-mentioned problems is the following a) to e);
a)Lactoferrin separated from skim milk, lactoferrin partially or fully saturated with iron, lactoferrin with iron removed, lactoferrin partially or completely saturated with metals other than iron (eg, copper, zinc, etc.) AntigenA plate preliminarily immobilized with a polyclonal antibody,
b)Claim 1A monoclonal antibody according to
c) Secondary antibody obtained by labeling an antibody against immunoglobulin of an immunized animal used for the preparation of the monoclonal antibody with an enzyme,
d) a substrate dye that develops color by the reaction of the enzyme; and
e) of standard concentrationA peptide comprising the amino acid sequence set forth in SEQ ID NO: 1A solution ofA peptide comprising the amino acid sequence set forth in SEQ ID NO: 1A reagent kit for detecting or quantifying and the following a) to g):
a)Lactoferrin separated from skim milk, lactoferrin partially or fully saturated with iron, lactoferrin with iron removed, lactoferrin partially or completely saturated with metals other than iron (eg, copper, zinc, etc.) AntigenA plate preliminarily immobilized with a polyclonal antibody,
b)Claim 1A monoclonal antibody according to
c) a secondary antibody in which an antibody against immunoglobulin of the immunized animal used for the preparation of the monoclonal antibody is labeled with biotin;
d) biotinylated alkaline phosphatase,
e) Streptavidin solution,
f) a substrate dye that develops color by the reaction of alkaline phosphatase, and
g) of standard concentrationA peptide comprising the amino acid sequence set forth in SEQ ID NO: 1A solution ofA peptide comprising the amino acid sequence set forth in SEQ ID NO: 1A reagent kit for detecting or quantifying is provided.
[0014]
  In the present invention, lactoferrins are natural lactoferrin,Specifically, lactoferrin separated from skim milk,It means lactoferrin partially or completely saturated with iron, lactoferrin from which iron has been removed, or lactoferrin partially or completely saturated with a metal other than iron (for example, copper, zinc, etc.).
[0015]
In the present specification, the percentage display is a value by weight unless otherwise specified.
Next, although the present invention will be described in detail, the second invention of the present invention will be described for easy understanding.
[0016]
<1> Hybridoma of the present invention
The second invention of the present invention produces a monoclonal antibody that specifically binds to a bovine or human lactoferrin fragment, for example, an antimicrobial peptide derived from bovine lactoferrin and an antimicrobial peptide derived from human lactoferrin. It relates to a hybridoma.
[0017]
In general, when an antibody against a peptide is prepared, it is desirable to use a continuous amino acid sequence of 5 residues or more in the amino acid sequence as an antigen. Therefore, for example, in the case of an antibacterial peptide derived from bovine lactoferrins, a peptide comprising an amino acid sequence of 5 or more residues among amino acid sequences corresponding to the 17th to 41st antibacterial peptides from the N-terminus of bovine lactoferrin is In the case of antibacterial peptides derived from a class, it is preferable to use, as an antigen, a peptide comprising an amino acid sequence of 5 residues or more in the amino acid sequence corresponding to the 47th human antibacterial peptide from the N-terminus of human lactoferrin.
[0018]
Examples of the fragments of bovine or human lactoferrin include peptides obtained by hydrolyzing lactoferrins with acid or enzymes, or peptides generated by digesting lactoferrins with enzymes in vivo.
[0019]
Hydrolysis with acid is performed by dissolving these lactoferrins in water or purified water or the like at a concentration of 0.1 to 20%, preferably 5 to 15%, and the resulting solution contains an inorganic salt such as hydrochloric acid or phosphoric acid, or An organic acid such as citric acid is added to adjust the pH of the solution to 1 to 4, desirably 2 to 3, and hydrolysis is carried out by heating at an appropriate temperature for a predetermined time according to the adjusted pH. For example, when the pH is adjusted to 1 to 2, it is 80 to 130 ° C, preferably 90 to 120 ° C. When the pH is adjusted to 2 to 4, it is 100 to 130 ° C, preferably 100 to 120 ° C. Heat for 1 to 120 minutes, preferably 5 to 60 minutes.
[0020]
When hydrolyzing with an enzyme, lactoferrins are dissolved in water, purified water or the like at a concentration of 0.5 to 20%, preferably 5 to 15%, and the obtained solution is used to obtain an optimum pH of the enzyme. Hydrolyze with The enzyme to be used is not particularly limited, and commercially available enzymes such as Morsin F (trademark, manufactured by Morishin Pharmaceutical Co., Ltd., optimal pH 2.5-3), porcine pepsin (manufactured by Wako Pure Chemical Industries, Ltd., optimal pH 2-3) Millisum AP (trademark, manufactured by Shin Nippon Chemical Co., Ltd., optimum pH 3.0), Amano M (trademark, produced by Amano Pharmaceutical Co., Ltd., optimum pH 3.0), Amano A (trademark, produced by Amano Pharmaceutical Co., Ltd., optimum pH 7.) 0), trypsin (manufactured by Novo Corp., optimum pH 8.0), etc. can be used alone or in combination.
[0021]
Peptides produced by enzymatic digestion of lactoferrin in vivo include, for example, antibacterial peptides; fragment peptides of lactoferrins that have been confirmed to exist in the feces, blood, and urine of infants (pediatric clinical, vol. 39 , Page 7, 1986).
[0022]
As a particularly desirable antibacterial peptide, peptides having the amino acid sequences described in SEQ ID NO: 1 to SEQ ID NO: 8 can be exemplified. Such a peptide can be purified from an enzymatic degradation product of lactoferrin by a known method (for example, JP-A-5-238948), but it can also be prepared by chemical synthesis such as liquid phase or solid phase synthesis method or recombinant DNA method. can do.
[0023]
In the case of producing an antibody, in the case of a peptide having about 20 or more amino acid residues, the known method (Shino Ohumi, “Anti-peptide antibody experimental protocol”, page 44, Shujunsha, 1994) Even if the animal is directly immunized as a peptide, it has sufficient antigenicity. However, in the case of peptides having about 5 to 20 amino acid residues, it is desirable to immunize by the above-mentioned known method in a state where these peptides are bound to a carrier protein. In this case, examples of the carrier protein include bovine serum albumin, keyhole limpet hemocyanin (hereinafter referred to as “KLH”), and the like. Bonding of these carrier proteins and peptides can be carried out by using m-maleimidobenzoyl N-hydroxysuccinide ester and covalently bonding the thiol group of the cysteine residue of the peptide and the amino group of the carrier protein. . As described above, a peptide-carrier protein complex that can be used for production of the antibody of the present invention is obtained.
[0024]
Using the above peptide or peptide-carrier protein complex as an antigen, mice were treated in a conventional manner [AM Campbell, Toshiaki Osawa, “Biochemical Experimental Method 10”, page 90, Tokyo Chemical Dojin, 1989]. The type of mouse to be used is not particularly limited, but generally a BALB / c mouse is used. The mice are immunized intraperitoneally or subcutaneously with Freund's complete or incomplete adjuvant, or intravenously or directly with no adjuvant, at a rate of 10-200 μg of antigen per mouse. Booster immunization is performed by the same method 2 to 3 weeks after the first immunization.
[0025]
Confirmation of the establishment of immunity is performed by collecting blood from mice and using the known enzyme-linked immunosorbent assay method [Biochimica et Biophysica Acta, Vol. 251, 427]. ~ 434 pages, 1971. Hereinafter, it is described as “ELISA method” by measuring the antibody titer in blood. It is possible to increase the probability of obtaining a hybridoma having antibody-producing activity by intravenously injecting an antigen into an immunized animal 3 days before cell fusion.
[0026]
Next, the spleen cells and myeloma cells of the mouse immunized as described above are fused. Examples of myeloma cells include BALB / c-derived X-63-Ag8-6.5.3 or SP2 / 0-Ag14.
[0027]
Examples of cell fusion and hybridoma selection methods are as follows. Polyethylene glycol having an average molecular weight of 1500 to 4000 is used as a cell fusion promoter, and spleen cells and myeloma cells are cultured in a commercially available hypoxanthine / aminopterin / thymidine medium (manufactured by Gibco, hereinafter referred to as “HAT medium”). Incubate. When a mutant strain in which hypoxanthine-guanine phosphoribosyltransferase gene or thymidine kinase gene is mutated is used as myeloma cells, cells after fusion treatment are cultured in this medium, unfused cells die, and only fused cells grow. So fused cells can be sorted out. Next, a hybridoma producing an antibody against the target antibacterial peptide by ELISA is selected as follows.
[0028]
A microplate used in the solid-phase ELISA method is coated with a lactoferrin fragment such as bovine antibacterial peptide or human antibacterial peptide, and the hybridoma culture supernatant is appropriately diluted and added to wash away unadsorbed substances. A secondary antibody conjugated with alkaline phosphatase or peroxidase is added. Unadsorbed secondary antibody was removed by washing, and paranitrophenyl phosphate, orthophenylenediamine or 2,2-azino-di- (3-ethylbenzthiazoline-6-sulfonic acid (manufactured by Bio-Rad) as a chromogenic substrate (Hereinafter referred to as “ABTS”) is added according to the enzyme used, and the well to which the culture supernatant of the cell producing the target antibody is added is colored. The hybridoma was cloned by the known limiting dilution method (AM Campbell, translated by Toshiaki Osawa, “Biochemical Experimental Method 10”, page 154, Tokyo Kagaku Dojin, 1989). A clone can be established, and the hybridoma of the second invention of the present invention can be obtained by the above operation.
[0029]
<2> Monoclonal antibody of the present invention
The first invention of the present invention relates to a monoclonal antibody that specifically binds to a bovine or human lactoferrin fragment, such as a bovine antimicrobial peptide or a human antimicrobial peptide.
[0030]
The monoclonal antibody according to the first invention of the present invention can be separated and purified from the hybridoma culture supernatant obtained as described above by a known ion exchange chromatography or protein A column. By inoculating a hybridoma into a mouse abdominal cavity and generating ascites, a monoclonal antibody concentrated about 1000 to 10,000 times can be obtained. This method is extremely efficient because a highly pure monoclonal antibody can be obtained simply by precipitation from ascites with ammonium sulfate.
[0031]
<3> Detection and quantification method of lactoferrin fragments
According to a third aspect of the present invention, a diluted sample containing a bovine or human lactoferrin fragment is immobilized on a solid phase, and the monoclonal antibody of the present invention is added to bind to the immobilized lactoferrin fragment. This method is a method for immunologically detecting or quantifying the fragments of the lactoferrins by detecting the monoclonal antibody obtained.
[0032]
Detection of the monoclonal antibody bound to the immobilized fragment of lactoferrin can be performed by using a labeled monoclonal antibody labeled with a labeling substance as the monoclonal antibody and detecting the labeling substance. In addition, after adding an unlabeled monoclonal antibody to the solid phase, a secondary antibody obtained by labeling an antibody against the immunoglobulin of the immunized animal used for the preparation of the monoclonal antibody with a labeling substance is added to the solid phase. The monoclonal antibody bound to the immobilized fragment of lactoferrin can also be indirectly detected by detecting the labeling substance.
[0033]
The secondary antibody is an antibody against the immunoglobulin of the immunized animal used for the preparation of the monoclonal antibody, and an anti-mouse immunoglobulin antibody is used for the mouse monoclonal antibody. In addition, if the class of the monoclonal antibody is IgG, the secondary antibody is preferably an anti-IgG antibody, but if it contains an anti-IgG antibody, it may contain antibodies against other classes of immunoglobulins.
[0034]
Examples of the solid phase for immobilizing the lactoferrin fragments include agarose beads, latex particles, polystyrene, nylon, and other microplates. Place a buffer solution containing a sample containing lactoferrin fragments on these solid phases, or immerse the solid phase in a buffer solution in which lactoferrin fragments are dissolved to immobilize the lactoferrin fragments on the solid phase. can do. Also, a lactoferrin fragment can be immobilized on a nitrocellulose filter or nylon membrane by dot blot or Western blot. Furthermore, when performing sandwich ELISA using two types of monoclonal antibodies that recognize different antigenic determinants, or a combination of monoclonal and polyclonal antibodies, one monoclonal antibody or polyclonal antibody is immobilized on a solid phase. The lactoferrin fragments can be indirectly immobilized on the solid phase by binding the lactoferrin fragments.
[0035]
Examples of labeling substances for labeling monoclonal antibodies include enzymes, biotin, fluorescent substances, luminescent substances, and radioisotopes. The enzyme can be detected by using a dye that develops color by an enzymatic reaction as a substrate. Such enzymes and substrates will be described later. The detection of biotin can be performed in the same manner as the detection of the enzyme by further binding an enzyme labeled with streptavidin to biotin, or by binding an enzyme biotinylated via streptavidin.
[0036]
Specific examples of detecting or quantifying bovine or human lactoferrin fragments, such as bovine antibacterial peptides or human antibacterial peptides, according to the method of the present invention are illustrated. In a 96-well (well) microplate well, a sample diluted with a protein adsorption buffer such as sodium carbonate-sodium bicarbonate buffer or phosphate buffer solution (hereinafter referred to as “PBS”) at pH 9.6 (for example, Lactoferrins, lactoferrin fragments, gastric juice, intestinal contents, stool, blood, urine, etc.) are added and adsorbed overnight at 4 ° C. or 1 hour at 37 ° C., and then the microplate is PBS-Tween (0. Washing is performed using a liquid in which Tween is mixed at a rate of 05%.
[0037]
Thereafter, each well may be blocked by adding a solution such as bovine serum albumin (BSA), gelatin, skim milk or the like in order to prevent non-specific adsorption of monoclonal antibodies and the like.
[0038]
Subsequently, the monoclonal antibody of the present invention is added as a primary antibody, reacted at 37 ° C. for 1 hour, the plate is washed in the same manner as described above, and an alkaline phosphatase-conjugated anti-mouse immunoglobulin antibody or peroxidase-conjugated anti-mouse immunity is used as a secondary antibody. Add globulin antibody and react at 37 ° C. for 1 hour. The plate was washed as described above, a chromogenic substrate was added, and the enzyme activity was measured by a conventional method [Bikchimica et Biophysica Acta, Vol. 251, pp. 427-434, 1971]. To do.
[0039]
When alkaline phosphatase is used, paranitrophenyl phosphate or the like can be exemplified as the chromogenic substrate, and when oxidase is used, orthophenylenediamine or ABTS can be exemplified as the chromogenic substrate.
[0040]
Further, in the detection method or quantification method of the present invention, instead of adding the secondary antibody, the step of binding the secondary antibody can be omitted by using the monoclonal antibody of the present invention directly labeled with an enzyme. .
[0041]
In addition, using a biotin-labeled anti-mouse immunoglobulin antibody as a secondary antibody, then adding streptavidin-conjugated alkaline phosphatase to form an avidin-biotin complex, and then adding a chromogenic substrate, the target peptide is highly sensitive. Can be detected or quantified. Furthermore, after adding the secondary antibody, a streptavidin solution may be added, and further biotinylated alkaline phosphatase may be added to allow alkaline phosphatase to bind to the secondary antibody via streptavidin.
[0042]
A sandwich ELISA method using two types of monoclonal antibodies recognizing different antigenic determinants, or a combination of the monoclonal antibody of the present invention and an anti-lactoferrin polyclonal antibody is particularly desirable from the viewpoints of quantification and detection sensitivity. In that case, one of the two types of monoclonal antibodies, or one of the monoclonal antibody and the polyclonal antibody, is immobilized on a solid phase, and a fragment of lactoferrins is bound thereto, and then the other antibody is used as described above. The same may be done. The anti-lactoferrin polyclonal antibody can be prepared by a known method in the same manner as the preparation of a normal polyclonal antibody. In addition, the lactoferrin may be sufficient as the antigen used for preparation of an anti- lactoferrin polyclonal antibody, and the fragment | piece of the lactoferrin of antimicrobial peptide may be sufficient as it.
[0043]
Furthermore, lactoferrin fragments can be detected and quantified by the so-called Western blot method using the monoclonal antibody of the present invention. For example, according to the method of Remli et al. [Nature, 227, 680-685, 1970], samples (lactoferrins, lactoferrin fragments, gastric juice, intestinal contents, stool, serum, urine, etc.) Are separated by polyacrylamide electrophoresis. After this gel is equilibrated with a transfer buffer, peptides and fragments in the gel are electrically transferred to a nitrocellulose membrane or a nylon filter and immobilized. The transferred membrane or filter is blocked with 10 mM Tris-HCl buffer, BSA, gelatin, skim milk or the like, and reacted with the monoclonal antibody of the present invention. Wash membrane or filter, add alkaline phosphatase-conjugated anti-mouse immunoglobulin antibody or peroxidase-conjugated anti-mouse immunoglobulin antibody as secondary antibody, wash, add chromogenic substrate, detect protein and peptide bound to antibody .
[0044]
When alkaline phosphatase is used as a chromogenic substrate, 5-bromo-4-chloro-3-indolyl phosphate, nitro blue tetrazolium, fast red, naphthol phosphate, etc. When peroxidase is used, 4-chloronaphthol , Diaminobenzidine, aminoethylcarbazole and the like.
[0045]
In addition, using a biotin-labeled anti-mouse immunoglobulin antibody as a secondary antibody, then adding streptavidin-conjugated alkaline phosphatase to form an avidin-biotin complex, and then adding a chromogenic substrate, the target peptide with higher sensitivity Fragments can also be detected.
[0046]
The color of the membrane or filter thus obtained can be measured with a densitometer, and the amount of antigen in the sample can be measured. It is also possible to use an antibody labeled with a radioisotope instead of an enzyme-labeled antibody or a biotin-labeled antibody. In this case, the radioactivity of a specific band on the autoradiography or membrane or filter is included. The membrane or filter can be cut out and measured with a liquid scintillator or the like.
[0047]
<4> Kit of the present invention
A fourth invention of the present invention is a reagent kit for detecting or quantifying bovine or human lactoferrin fragments (hereinafter referred to as kit), a plate on which an anti-lactoferrin polyclonal antibody is immobilized in advance, the present invention Monoclonal antibody, a secondary antibody obtained by labeling an antibody against immunoglobulin of an immunized animal used for the preparation of the monoclonal antibody with a labeling substance, a reagent for detecting the labeling substance, and human or bovine lactoferrin at a standard concentration A solution of fragments.
[0048]
In the case where an enzyme-labeled antibody is used as the labeled secondary antibody, the kit contains a substrate dye that develops color by the reaction of the enzyme as a reagent for detecting the labeling substance. In addition, when a biotin-labeled antibody is used as the secondary antibody, a streptavidin-binding enzyme or a biotinylated enzyme-streptavidin solution as a reagent for detecting the labeling substance, and a substrate dye that develops color by the reaction of the enzyme Is included. When a radiolabeled antibody is used as the labeled secondary antibody, the label can be detected by an X-ray film.
[0049]
When the monoclonal antibody of the present invention is derived from a mouse cell, specifically, an alkaline phosphatase-labeled mouse immunoglobulin antibody, a peroxidase-labeled anti-mouse immunoglobulin antibody, a biotinylated anti-mouse immunoglobulin antibody as an enzyme-labeled secondary antibody,¹²⁵Examples include I-labeled anti-mouse immunoglobulin antibody.
[0050]
Each reagent constituting the kit of the present invention is individually packaged to form one set. In addition to the above, a washing buffer solution, a diluting solution, and other substrates for coloring the substrate dye may be included.
[0051]
Next, a test example is shown and this invention is explained in full detail.
[0052]
[Test Example 1]
This test was conducted to examine the specificity of the obtained monoclonal antibody.
[0053]
1) Sample preparation
An antibacterial peptide was prepared by the same method as Reference Example 2 described later. A monoclonal antibody was prepared by the same method as in Example 1 described later.
[0054]
2) Test method
Lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd., purity 90%), antibacterial peptide or bovine serum albumin (manufactured by Sigma Co., hereinafter referred to as “BSA”) is dissolved in 1 ml of PBS solution at a rate of 0.1 mg. Added to well microplate and allowed to bind for 1 hour at 37 ° C. The plate in which the antigen was bound to each well was washed with a PBS-Tween solution, and 150 μl of a PBS solution containing 1% gelatin was added to each well and blocked at 37 ° C. for 1 hour. The plate was washed in the same manner as described above, and the monoclonal antibody and the control rabbit anti-bovine lactoferrin antibody (manufactured by Purcell) were diluted appropriately, 100 μl was added to each well, and the antibody was allowed to bind at 37 ° C. for 1 hour. The plate was washed in the same manner as described above, and an alkaline phosphatase-labeled anti-mouse immunoglobulin antibody (Zymed) or an alkaline phosphatase-labeled anti-rabbit immunoglobulin antibody (Zymed) was appropriately diluted as a secondary antibody in each well. 100 μl was added and reacted at 37 ° C. for 1 hour.
[0055]
Thereafter, p-nitrophenyl sodium phosphate (manufactured by Bio-Rad) was added to the wells washed as described above as a chromogenic substrate, and after 30 minutes, the absorbance at 405 nm was measured to test the enzyme activity.
[0056]
3) Test results
The results of this test are as shown in Table 1. As is apparent from Table 1, it was confirmed that the monoclonal antibody of the present invention did not bind to natural lactoferrin and specifically bound only to the antibacterial peptide. In addition, although it tested by changing the kind of monoclonal antibody and antibacterial peptide, the substantially same result was obtained.
[0057]
[Table 1]

[0058]
[Test Example 2]
This test was conducted in order to examine the cross-reactivity of the protein or antibacterial peptide modified with SDS (sodium dodecyl sulfate) and 2-mercaptoethanol and the antibody.
[0059]
1) Sample preparation
Bovine lactoferrin and antibacterial peptide were prepared by the same method as in Reference Example 1 and Reference Example 2. A monoclonal antibody was prepared by the same method as in Example 1.
[0060]
2) Test method
Lactoferrin and antibacterial peptide are mixed with 50 mM Tris-HCl buffer (pH 6.8), 1% SDS (manufactured by Nacalai Tesque), 0.05% bromophenol blue (manufactured by Wako Pure Chemical Industries), 5% glycerol (Nacalai Tesque) SDS-polyacrylamide gel (dissolved in 1% 2-mercaptoethanol (manufactured by Bio-Rad)), heated at 100 ° C. for 2 minutes to be denatured, and using a discontinuous buffer solution by the method of Remli et al. 15%) and separated by electrophoresis.
[0061]
Proteins and peptides separated on the electrophoresis gel were electrically blotted onto a nitrocellulose sheet having a pore size of 0.25 μm using a Trans Blot transfer apparatus (Bio-Rad) according to the procedure recommended by the company. The blotted nitrocellulose sheet was blocked with a mixture of 1% BSA, 10 mM Tris-HCl buffer and 50 mM sodium chloride (hereinafter referred to as “T10N50 solution”) while gently shaking for 2 hours.
[0062]
Thereafter, the nitrocellulose sheet was shaken in a 1% BSA, T10N50 solution containing the monoclonal antibody of the present invention for 1 hour to bind the antibody. This nitrocellulose sheet was thoroughly washed with 10 mM Tris-HCl buffer, 50 mM sodium chloride and 0.05% nonidet P-40 (hereinafter referred to as “T10N50NP40 solution”), and then 5 μCi.¹²⁵Shake for 1 hour in a solution containing I-labeled rabbit anti-mouse IgG (Amersham) and 1% BSA and T10N50 solution, wash with T10N50NP40 solution, dry, and expose to film (Kodak XAR-5). It was.
[0063]
3) Test results
The result of this test is as shown in FIG. FIG. 1 is an amide black staining diagram (A in FIG. 1) of a protein blotted on nitrocellulose paper and an immunostaining diagram (B in FIG. 1) using the monoclonal antibody of the present invention. Molecular weight markers, bovine lactoferrin and antimicrobial peptides are shown.
[0064]
As is apparent from FIG. 1, the antibody of the present invention not only binds to the antibacterial peptide but also binds to lactoferrin that has been heat-denatured with SDS and 2-mercaptoethanol. In addition, although it tested by changing the kind of monoclonal antibody and antibacterial peptide, the substantially same result was obtained.
[0065]
From Test Example 1 and Test Example 2, it is clear that the antibody of the present invention does not bind to natural lactoferrin but specifically binds to an antimicrobial peptide.
[0066]
[Reference Example 1]
An ion exchanger (CM-Sepharose FF (Pharmacia)) was packed in a 3 L column, 4 L of 100 mM hydrochloric acid was passed through, washed with water, and the ion exchanger was equilibrated. 22 L of human skim milk of pH 6.9 cooled to 4 ° C. was passed through the column at a flow rate of 500 ml / min, the permeate was collected, and again passed through the column in the same manner. Next, distilled water was passed at a flow rate of 500 ml / min, and 4 L of 10% saline was passed at a flow rate of 2 L / min to obtain 3.5 L of a basic protein solution adsorbed on the ion exchanger.
[0067]
To this recovered solution, ammonium sulfate is added to a saturation degree of 80%, the protein is precipitated, and centrifuged (3000 × g) to recover the precipitate, washed with an ammonium sulfate solution with a saturation degree of 80%, and removed. 200 ml of ionic water is added to dissolve, and the solution is ultrafiltered using an ultrafiltration membrane module (manufactured by Asahi Kasei Co., Ltd. SLP0053), water is added, and diafiltration is performed using the same apparatus. , Desalted and lyophilized to obtain about 17 g of powdered human lactoferrin.
[0068]
The resulting human lactoferrin lyophilized product was separated by electrophoresis, and the purity was measured. As a result, it was about 97%.
[0069]
[Reference Example 2]
10 g of bovine lactoferrin (manufactured by Morinaga Milk Industry Co., Ltd., purity: about 90%) separated from skim milk is dissolved in distilled water at a concentration of 5% (w / v), and 1N hydrochloric acid is added to adjust the pH to 3.0. Then, pepsin (manufactured by Wako Pure Chemical Industries, Ltd.) was added at a ratio of 3% of the substrate, hydrolyzed at 37 ° C. for 4 hours, then heated to 80 ° C. for 15 minutes to deactivate pepsin, and 1N hydroxylation Sodium was added to adjust the pH to 7.0, centrifugation was performed at 1500 × g for 30 minutes to remove insoluble matters, and lyophilization was performed to obtain about 7.9 g of a powdery peptide mixture containing an antibacterial peptide. .
[0070]
25 ml of cation exchanger (Carboxymethyl Toyopearl (trademark; manufactured by Tosoh Corp., 650M)) was thoroughly washed with 100 mM sodium monophosphate-dibasic sodium phosphate buffer (pH 7.8) to obtain an equilibrated slurry. The antibacterial peptide mixture solution dissolved in the buffer solution was added and sufficiently adsorbed while stirring with a magnetic stirrer in a beaker. A column (5 cm in length, 3 cm in diameter) is packed with a cation exchanger adsorbing an antibacterial peptide mixture, and the buffer solution is used at a flow rate of 5 ml / min until the absorbance at 280 nm of the washing solution is 0.03 or less. Washed. Similarly, 100 mM sodium chloride, 100 mM sodium monophosphate-dibasic sodium phosphate buffer (pH 7.8) was passed through the column at a flow rate of 5 ml / min until the absorbance at 280 nm of the washing solution reached 0.03 or less. The peptide adsorbed non-specifically to the gel was washed.
[0071]
Next, 2M ammonium acetate (pH 7.8) was passed through the column to obtain 10 ml of a solution containing an antibacterial peptide. The obtained antibacterial peptide solution was injected into a PD10 column for desalting (Pharmacia) equilibrated with water in five portions to obtain a desalted antibacterial peptide solution, which was freeze-dried. About 18 mg of powdery antibacterial peptide was obtained.
[0072]
The obtained purified peptide was confirmed to have the amino acid sequence shown in SEQ ID NO: 1 by conventional amino acid analysis, amino acid sequence analysis, elemental analysis and mass spectrometry. In SEQ ID NO: 1, it is presumed that the third Cys and the 21st Cys are disulfide bonded.
[0073]
[Reference Example 3]
Using a peptide synthesizer (Pharmacia LKB Biotechnology Co., Ltd. LKB Biolynx 4170) according to the method of Shepard et al. (Journal of Chemical Society Perkin I, page 538, 1981) Based on the solid phase peptide synthesis method, a peptide having the sequence shown in SEQ ID NO: 2 was synthesized as follows.
[0074]
NovaSynKA derivative resin (Fmoc-Cys (Acm) -NovaSynKA. Calbiochem-Novabiochem) 0.1 meq (1 g) was used to repeat the deprotection reaction and condensation reaction according to the peptide synthesizer synthesis program. Extended. That is, 9-fluorenylmethoxycarbonyl (hereinafter referred to as “Fmoc”) group which is an amino protecting group is cleaved and removed by 20% piperidine / dimethylformamide (manufactured by Kanto Chemical Co., Inc., hereinafter referred to as “DMF”). Then, washing with DMF, and subsequent reaction of 0.5 mmol each of Fmoc-amino acid active ester / N-hydroxybenzotriazole (hereinafter referred to as “HOBt”) and washing with DMF were repeated.
After the condensation reaction, a Kaiser test was performed as necessary to confirm that the coupling was complete. A 0.5 mmol cartridge was used for the synthesis. After all the peptide chain elongation reactions were completed, the Fmoc group was cleaved with 20% piperidine / DMF, washed with DMF, and acetylated with 10% acetic anhydride / DMF. After confirming the completion of acetylation by the Kaiser test, the resin was treated with DMF, tert-pentyl alcohol (manufactured by Kanto Chemical Co., Inc.), acetic acid (manufactured by Kanto Chemical Co., Inc.), tert-pentyl alcohol (manufactured by Kanto Chemical Co., Inc.), DMF, Diethyl ether (manufactured by Kokusan Chemical Co., Ltd.) was washed in order and dried in vacuum.
[0075]
To 650 mg of the above-mentioned protected peptide resin, 1.0 ml of ethanedithiol (manufactured by Watanabe Chemical Co., Ltd.), 200 ml of m-cresol (manufactured by Watanabe Chemical Co., Ltd.) and 2.4 ml of thioanisole (manufactured by Watanabe Chemical Co., Ltd.) are allowed to stand at room temperature for 15 minutes under an argon stream. The mixture was stirred and then further stirred for 10 minutes under ice cooling. To this, 15 ml of trifluoroacetic acid (manufactured by Watanabe Chemical Co., Ltd., hereinafter referred to as “TFA”) was added and stirred for 10 minutes, and 2.6 ml of trimethylsilyl bromide (manufactured by Watanabe Chemical Co., Ltd.) was added and stirred for 50 minutes. The resin was removed by filtration through a glass filter, and the filtrate was immediately concentrated under reduced pressure. Add pre-cooled diethyl ether (made by Kokusan Chemical Co., Ltd.) to the residue, transfer to a centrifuge tube, centrifuge (2500 rpm for 5 minutes), discard the supernatant, add cold diethyl ether anew and stir well. The operation of centrifugation again was repeated 4 times. The peptide precipitate was vacuum dried, dissolved in water and lyophilized to obtain about 94.3 mg of crude peptide.
[0076]
CyBF (9cm) peptide 94.3mg AgBF_Four(10 eq, manufactured by Watanabe Chemical Co., Ltd.) / Anisole (10 eq, manufactured by Watanabe Chemical Co., Ltd.) / TFA solution was added and stirred at 4 ° C. for 60 minutes. And vacuum-dried SHBaseFree peptide was obtained. 50% DMSO (manufactured by Watanabe Chemical Co.) / 1N HCl (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the SH group free peptide and stirred at room temperature for 7 hours. The disulfide production was monitored by high performance liquid chromatography (HPLC). Add water and supply it to a synthetic adsorbent HP20 (Mitsubishi Chemical Corporation) column to adsorb, wash thoroughly with water, 60% CH_ThreeThe peptide was eluted with CN / 1N AcOH (manufactured by Kanto Chemical Co., Inc.), concentrated by centrifugation, freeze-dried with water, and 61.1 mg of crude peptide was obtained.
[0077]
The crude peptide was dissolved in water, centrifuged (15000 rpm for 5 minutes), the supernatant was filtered through a 0.45 mm filter, and this solution was subjected to high performance liquid chromatography (HPLC) to purify the peptide. For the HPLC, an LKB 2151 high-pressure gradient system (Pharmacia) was used, and for the column, a reverse phase commercial column (Tosoh, ODS120T, 21.5 × 300 mm) was used. The eluent was 0.1% TFA / water solution A and 80% acetonitrile / water solution B was eluted with a concentration linear gradient from solution A to solution B. The chromatogram was recognized as a single peak, and the corresponding fraction was collected. This preparative operation was repeated several times and freeze-dried to obtain about 30.5 mg of purified antibacterial peptide.
[0078]
The purified peptide was subjected to conventional amino acid analysis, amino acid sequence analysis, elemental analysis, and mass spectrometry, and confirmed to have the target amino acid sequence of SEQ ID NO: 2.
[0079]
[Reference Example 4]
A peptide having the amino acid sequence shown in SEQ ID NO: 3 was synthesized in the same manner as in Reference Example 3 except that the order of amino acids used was different. The yield of the acylated protected peptide resin was about 581 mg, and the yield of the crude peptide after deprotection and deresining was about 83.3 mg, and about 59.9 mg of purified antimicrobial peptide was obtained. It was confirmed to have the amino acid sequence described in SEQ ID NO: 3 by the same test method as in Reference Example 3.
[0080]
[Reference Example 5]
The same operation as in Reference Example 2 was performed using 10 g (purity 97%) of human lactoferrin purified in the same manner as in Reference Example 1. The yield of antibacterial peptide after desalting was about 25 mg.
[0081]
The obtained purified peptide was confirmed to be a dipeptide having the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5 by conventional amino acid analysis, amino acid sequence analysis, elemental analysis, and mass spectrometry. The 9th Cys and 26th Cys of the peptide shown in SEQ ID NO: 4 are disulfide bonded, and the 35th Cys of the peptide shown in SEQ ID NO: 4 and the 10th Cys of the peptide shown in SEQ ID NO: 5 are disulfide bonded. It is estimated that
[0082]
[Reference Example 6]
A peptide having the amino acid sequence shown in SEQ ID NO: 6 was synthesized in the same manner as in Reference Example 3 except that the order of amino acids used was different. The yield of acylated protected peptide resin was about 830 mg, the yield of Cys (Acm) crude peptide after deprotection and deresin was about 201.0 mg, oxidized crude peptide 169.2 mg, and purified antimicrobial peptide about 62.1 mg. Obtained. It was confirmed to have the amino acid sequence described in SEQ ID NO: 6 by the same test method as in Reference Example 3.
[0083]
[Reference Example 7]
A peptide having the amino acid sequence shown in SEQ ID NO: 7 was synthesized in the same manner as in Reference Example 3 except that the order of amino acids used was different. The yield of the acylated protected peptide resin was about 570 mg, and the yield of the crude peptide after deprotection and deresining was about 59.5 mg, and about 38.2 mg of purified antimicrobial peptide was obtained. It was confirmed to have the amino acid sequence described in SEQ ID NO: 7 by the same test method as in Reference Example 3.
[0084]
[Reference Example 8]
A peptide having the amino acid sequence shown in SEQ ID NO: 8 was synthesized in the same manner as in Reference Example 3 except that the order of amino acids used was different. The yield of acylated protected peptide resin was about 750 mg, the yield of Cys (Acm) crude peptide after deprotection and deresining was about 186.8 mg, 131.2 mg of oxidized crude peptide, about 51. 1 of purified antimicrobial peptide. 4 mg was obtained. It was confirmed to have the amino acid sequence described in SEQ ID NO: 8 by the same test method as in Reference Example 3.
[0085]
[Reference Example 9]
This example shows the preparation of a polyclonal antibody contained in the kit of the present invention for quantifying peptides derived from human lactoferrins.
[0086]
400 μg of the antibacterial peptide dimer having the amino acid sequence of SEQ ID NO: 4 and SEQ ID NO: 5 produced by the same method as in Reference Example 5 was dissolved in 1.2 ml of physiological saline, together with 1.5 ml of Freund's complete adjuvant, Four 14-week-old Jla: JW male rabbits were injected subcutaneously with 0.5 ml per mouse. The antibacterial peptide dimer solution prepared in the same manner as described above was injected together with Freund's incomplete adjuvant at 3 weeks and 5 weeks after the initial immunization. Seven days after the final immunization, the rabbits were sacrificed and serum was prepared. 150 ml of serum was obtained per animal.
[0087]
A column (20 mm × 800 mm) packed with 25 ml of Protein A gel (Biorad) equilibrated with MAPSII binding buffer (BioRad) was diluted 5 times with MAPSII binding buffer to 5 ml / min. Each was applied at a flow rate. The column was washed with 400 ml of MAPSII binding buffer, and then the bound antibody was eluted with 70 ml of MAPSII elution buffer (Biorad). The antibody solution immediately adjusted to neutral pH with sodium hydroxide was dispensed and stored frozen until use.
[0088]
【Example】
EXAMPLES Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.
[0089]
[Example 1]
This example shows the production of an anti-bovine antibacterial peptide monoclonal antibody-producing hybridoma using a synthetic peptide as an antigen.
[0090]
(i) Preparation of antigen
16 mg of KLH (Pierce) was dissolved in 1 ml of 10 mM sodium phosphate buffer (pH 7.2), and 2.8 mg of m-maleimidobenzoyl-N-hydrosuccinimide ester (m-maleimidobenzoyl-N- hydrosuccinimide ester: hereinafter referred to as “MBS” (manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred for 30 minutes. The solution was filtered with a 0.45 μm filter and subjected to Sephadex G-25 (Sephadex G-25: Pharmacia) column chromatography to collect about 5 ml of KLH-MBS solution.
[0091]
To this solution was added 2.5 ml of 0.2 M disodium phosphate solution and 10 mg of a chemically synthesized antibacterial peptide (having the sequence shown in SEQ ID NO: 2) produced by the same method as in Reference Example 3, and argon gas was used. The solution was replaced, reacted at room temperature for 30 minutes, and this solution was stored frozen until used for immunization.
[0092]
(ii) Mouse sensitization
50 μl of the above solution was mixed with 1 ml of physiological saline, and 400 μl per mouse was injected subcutaneously into 5 6-week-old BALB / c mice with 1.5 ml of Freund's complete adjuvant. Thereafter, immunization was carried out three times in the same manner at intervals of 2 weeks. In the course of immunization, the increase in the antibody titer of serum against the antigen was collected from mice and observed by ELISA. The mouse with the highest antibody titer was injected with 15 μg of the chemically synthesized antibacterial peptide shown in SEQ ID NO: 2 dissolved in 150 μl of PBS through the tail vein to give a boost.
[0093]
(Iii) Cell fusion
Three days after the final immunization, splenocytes were aseptically removed, loosened into single cells with a stainless mesh, washed 3 times with RPMI 1640 (25 mM HEPES and 2 mM L-glutamine), and about 1/4 volume of splenocytes X-6-3 The mixture was mixed with Ag86.5.3 myeloma cells (Gibco) and centrifuged. 50% polyethylene glycol (PEG) 1500 was added to the cell pellet, cell fusion was performed, and the centrifuged cell pellet was subjected to hypoxanthine thymidine (hypoxantine) containing 30% fetal calf serum (Gibco, hereinafter referred to as FCS). thymidine) medium was added, seeded on 19 96-well microplates, and HAT medium was added to each well the next day.
[0094]
(Iv) Limit dilution and antibody specificity
Colonies grew from 1300 wells after the fusion, and these culture supernatants were subjected to ELISA using an antibacterial peptide derived from bovine lactoferrin as an antigen to obtain hybridomas that strongly reacted with the antigen. Cloning by limiting dilution was repeated 4 times to obtain 8 clones showing stable antibody production.
[0095]
[Example 2]
This example shows the production of a hybridoma producing an anti-bovine antibacterial peptide monoclonal antibody using a bovine antibacterial peptide prepared from bovine lactoferrin as an antigen.
[0096]
1 mg of an antibacterial peptide derived from bovine lactoferrin shown in SEQ ID NO: 1 produced by the same method as in Reference Example 2 was dissolved in 1 ml of physiological saline and 8 6-week-old BALB / c together with 1.5 ml of Freund's complete adjuvant. Mice were injected subcutaneously at a rate of 300 μl per mouse. Thereafter, 12 clones showing stable antibody production were obtained by the same method as in Example 1.
[0097]
[Example 3]
This example shows the preparation of a hybridoma producing an anti-human antibacterial peptide monoclonal antibody using a synthetic peptide as an antigen.
[0098]
Six clones producing antibodies were obtained by the same method as in Example 2 except that the antimicrobial peptide shown in SEQ ID NO: 6 produced by the same method as in Reference Example 6 was used.
[0099]
[Example 4]
Eight clones producing antibodies were obtained by the same method as in Example 1 except that the antibacterial peptide shown in SEQ ID NO: 3 produced by the same method as in Reference Example 4 was used.
[0100]
[Example 5]
Ten clones producing antibodies were obtained by the same method as in Example 2 except that the antimicrobial peptide dimer shown in SEQ ID NO: 4 and SEQ ID NO: 5 produced by the same method as in Reference Example 5 was used.
[0101]
[Example 6]
Eight clones producing antibodies were obtained by the same method as in Example 1 except that the antibacterial peptide shown in SEQ ID NO: 7 produced by the same method as in Reference Example 7 was used.
[0102]
[Example 7]
Six clones producing antibodies were obtained by the same method as in Example 2 except that the antibacterial peptide shown in SEQ ID NO: 8 produced by the same method as in Reference Example 8 was used.
[0103]
[Example 8]
This example shows a method for quantifying an antimicrobial peptide derived from bovine lactoferrin.
[0104]
Human gastric juice taken orally with 100 ml of a solution containing 1 mg of bovine lactoferrin per ml was collected 5 minutes later, and pepstatin A (manufactured by Sigma) was added to the collected gastric juice at a rate of 0.01%. Protein degradation due to protease activity was prevented.
[0105]
A gastric juice sample and a standard antibacterial peptide were separated by electrophoresis by the same method as in Test Example 2, blotted on a membrane, and immunostained using an antibody produced by a clone obtained by the same method as in Example 1. .
[0106]
As a result of quantifying the band of the immunostained antibacterial peptide with the PDI electrophoresis analysis system (manufactured by Toyobo Co., Ltd.), the active antibacterial peptide generated from the ingested bovine lactoferrin was found in the gastric juice of the ingested bovine lactoferrin. It was found to be present and its concentration was 20 μg / ml.
[0107]
[Example 9]
This example shows a reagent kit for detecting or quantifying antibacterial peptides.
[0108]
Using the monoclonal antibody according to the present invention, a kit for detecting or quantifying human antibacterial peptides in gastric juice, intestinal contents, stool, blood and urine by ELISA was prepared as follows.
[0109]
The kit of the present invention comprises an anti-human antibacterial peptide polyclonal antibody pre-coated 96-well microplate produced by the same method as in Reference Example 9, a phosphate buffer, and an anti-human antibacterial peptide monoclonal produced by the same method as in Example 5. The antibody, washing solution, peroxidase-conjugated anti-mouse antibody, human antibacterial peptide standard solution, and 0.1% o-phenylenediamine solution are contained individually.
[0110]
【The invention's effect】
As described in detail above, the present invention relates to a monoclonal antibody that binds to a fragment of bovine or human lactoferrin, such as an antibacterial peptide, and does not bind to natural lactoferrin, a hybridoma that produces the antibody, bovine or human lactoferrin. The present invention relates to a method for detecting or quantifying a class of fragments and a reagent kit used for the method for detecting or quantifying, and the effects produced by the present invention are as follows.
[0111]
1) An antibody highly specific to a fragment of human or bovine derived lactoferrin and a cell line producing the same are obtained.
2) Specific use of lactoferrin-derived antibacterial peptides contained in human or bovine-derived lactoferrins, such as gastric juice, intestinal contents, feces, blood, urine, etc., without using complicated devices And can be detected or quantified with high sensitivity.
[0112]
[Sequence Listing]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[Brief description of the drawings]
FIG. 1 is an amide black staining diagram (A in FIG. 1) and an immunostaining diagram using the monoclonal antibody of the present invention (B in FIG. 1) of nitrocellulose paper blotted with proteins after electrophoresis.

Claims (6)

Lactoferrin , which is a monoclonal antibody using a chemically synthesized antibacterial peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 as an antigen, which binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and is separated from skim milk Monoclonal antibody that does not bind to. Lactoferrin , which is a monoclonal antibody using a chemically synthesized antibacterial peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 as an antigen, which binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and is separated from skim milk A hybridoma that produces a monoclonal antibody that does not bind to. A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 contained in a sample is immunologically measured using the monoclonal antibody described in claim 1 from the amino acid sequence shown in SEQ ID NO: 1. A method for detecting or quantifying the peptide . Next a) or a);
A) A diluted sample containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 is immobilized on a solid phase, the monoclonal antibody of claim 1 is added, and the immunoglobulin of the immunized animal used for the preparation of the monoclonal antibody A secondary antibody labeled with a labeling substance with an antibody against is added to the solid phase, and the labeling substance is detected;
A) A diluted sample containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 is immobilized on a solid phase, a labeled monoclonal antibody obtained by labeling the monoclonal antibody of claim 1 with a labeling substance is added, and this label is added. A method for detecting or quantifying a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, comprising any one of detecting a substance. The following a) to e);
a) Any of lactoferrin separated from skim milk, lactoferrin partially or completely saturated with iron, lactoferrin from which iron has been removed, or lactoferrin partially or completely saturated with metals other than iron (eg, copper, zinc, etc.) A plate preliminarily immobilized with a polyclonal antibody using the
b) the monoclonal antibody of claim 1 ,
c) a secondary antibody obtained by labeling an antibody against immunoglobulin of the immunized animal used for the preparation of the monoclonal antibody with an enzyme ,
d) detecting or quantifying a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 comprising a substrate dye that develops color by the reaction of the enzyme, and e) a solution of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 at a standard concentration. Reagent kit to do. The following a) to g);
a) Any of lactoferrin separated from skim milk, lactoferrin partially or completely saturated with iron, lactoferrin from which iron has been removed, or lactoferrin partially or completely saturated with metals other than iron (eg, copper, zinc, etc.) A plate preliminarily immobilized with a polyclonal antibody using the
b) the monoclonal antibody of claim 1 ,
c) a secondary antibody in which an antibody against immunoglobulin of the immunized animal used for the preparation of the monoclonal antibody is labeled with biotin;
d) biotinylated alkaline phosphatase,
e) Streptavidin solution,
f) detecting or quantifying a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, comprising a substrate dye that develops color by the reaction of alkaline phosphatase, and g) a solution of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 at a standard concentration. Reagent kit to do.

Images