JP2005172637A - ポリペプチドとレセプターとの相互作用を検出する方法、該検出する方法を用いてリガンドまたはリガンド変異体をスクリーニングする方法および該検出する方法を用いる診断方法 - Google Patents
ポリペプチドとレセプターとの相互作用を検出する方法、該検出する方法を用いてリガンドまたはリガンド変異体をスクリーニングする方法および該検出する方法を用いる診断方法 Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
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Abstract
【解決手段】 担体上にポリペプチドを固定化し、これに膜レセプターにおける細胞外ドメインを含むが膜結合に必須の領域を含まないレセプター誘導体を反応させ、固定化したポリペプチドとレセプター誘導体との相互作用を検出することにより、ポリペプチドとレセプターとの相互作用を検出する方法。
【選択図】 図1
Description
(1)担体上にポリペプチドを固定化し、これに膜レセプターにおける細胞外ドメインを含むが膜結合に必須の領域を含まないレセプター誘導体を反応させ、固定化したポリペプチドとレセプター誘導体との相互作用を検出することにより、ポリペプチドとレセプターとの相互作用を検出する方法。
(2)担体が、基板上にポリペプチドと共有結合しうる官能基、ならびにダイヤモンド、軟ダイヤモンド、炭素系物質および炭化物から選ばれる少なくとも1種の表面層を有する固体支持体である、(1)記載の方法。
(3)ポリペプチドおよび多価アルコールを含むスポッティング溶液を担体上にスポッティングすることによりポリペプチドを担体上に固定化する(1)または(2)記載の方法。
(4)担体上に固定化するポリペプチドが、膜レセプターに対するリガンドまたはリガンド変異体である(1)〜(3)のいずれかに記載の方法。
(5)(4)記載の検出方法を用いて、膜レセプターに対するリガンドの変異体をスクリーニングする方法。
(6)(1)〜(3)のいずれかに記載の検出方法を用いて、膜レセプターに対するリガンドをスクリーニングする方法。
(7)(1)〜(4)のいずれかに記載の検出方法を用いる診断方法。
本発明で対象とする膜レセプターとしては、ペプチドホルモン、神経伝達物質、増殖因子、サイトカイン、カテコールアミンなどに対するレセプターが挙げられ、機能的には3量体Gタンパク共役型レセプター、イオンチャンネル共役型レセプター、プロテインキナーゼ型レセプター、介助レセプター(co-receptor)に分類される。また、形質膜を1回貫通するレセプター、形質膜を4回貫通するレセプターおよび形質膜を7回貫通するレセプター、さらに脂質を介して膜に結合するGPIアンカー型レセプターに分類することもでき、本発明は、形質膜を1回貫通するレセプターやGPIアンカー型レセプターとポリペプチドの相互作用の検出に好適である。具体的にはEGFレセプター、PDGFレセプター、アデノシンレセプター、FGFレセプター、TGFβレセプター、インシュリンレセプター、IGF-Iレセプター、アンジオテンシンレセプター、OBレセプター、メラノコルチンレセプター、アドレナリンレセプター、トロンビンレセプター、オキシトシンレセプター、イノシトール3リン酸レセプター、FSHレセプター、TSHレセプター、インターフェロンレセプター、インターロイキンレセプター、G-CSFレセプター、ケモカインレセプター、チロシンキナーゼレセプター、GDNFレセプター、TNFレセプター、グリピカンなどが挙げられる。
本発明においては、担体にポリペプチドを固定化し、該ポリペプチドに可溶性レセプター誘導体を反応させる。担体に固定化するポリペプチドとしては、膜レセプターと相互作用する可能性のあるポリペプチドであればいずれも対象となりうる。天然に存在するポリペプチドでもよいし、人工的に合成したものでもよい。本発明においてポリペプチドには、オリゴペプチド、タンパク質およびタンパク質断片等も包含される。
ポリペプチドを固定化する担体としては、当技術分野で通常用いられるものを使用できる。例えば、白金、白金黒、金、パラジウム、ロジウム、銀、水銀、タングステンおよびそれらの化合物などの貴金属、およびグラファイト、カーボンファイバーに代表される炭素などの導電体材料;単結晶シリコン、アモルファスシリコン、炭化ケイ素、酸化ケイ素、窒化ケイ素などに代表される半導体材料、SOI(シリコン・オン・インシュレータ)などに代表されるこれら半導体材料の複合素材;ガラス、石英ガラス、アルミナ、サファイア、セラミクス、フォルステライト、感光性ガラスなどの無機材料;ポリエチレン、エチレン、ポリプロビレン、ポリイソブチレン、ポリエチレンテレフタレート、不飽和ポリエステル、含フッ素樹脂、ポリ塩化ビニル、ポリ塩化ビニリデン、ポリ酢酸ビニル、ポリビニルアルコール、ポリビニルアセタール、アクリル樹脂、ポリアクリロニトリル、ポリスチレン、アセタール樹脂、ポリカーボネート、ポリアミド、フェノール樹脂、ユリア樹脂、エポキシ樹脂、メラミン樹脂、スチレン・アクリロニトリル共重合体、アクリロニトリル・ブタジエンスチレン共重合体、ポリフェニレンオキサイドおよびポリスルホンなどの有機材料等が挙げられる。
官能基としてアミノ基を導入するために用いられる化合物としては、例えばアミノ酸が挙げられる。
次に、ポリペプチドを固定化した担体に、可溶性レセプター誘導体を反応させ、固定化したポリペプチドと可溶性レセプター誘導体との相互作用を検出する方法について説明する。
1-1. ErbB1細胞外ドメイン(ErbB1 ECD)-FLAG
pCO12-EGFR保持菌体を白金耳でとり50μg/mlアンピシリンを含むLB寒天培地(LB-Amp50 Agar)にストリークしコロニーアイソレイションを行った。次に50μg/mlアンピシリンを含むLB培地(LB-Amp50)5mlに植菌し、37℃で一晩、280/分で振盪培養した。培養した大腸菌を室温で3000rpm、10分間遠心し集菌してQIAprep Spin Miniprep Kit(QIAGEN)でプラスミドを抽出した。このプラスミド溶液を1μl(144ng相当)を0.2mlチューブに採り、2.5mM dNTP(TaKaRa)を6μl、10×PFUバッファを5μl、10μMフォワードプライマー♯530(配列番号1)と10μMリバースプライマー♯531(配列番号2)をそれぞれ1μlずつ、滅菌Ultra Pure Water(UPW)を35.5μl加え最後にPfu Turbo DNA polymerase(STRATAGENE)を0.5μl加えてサーマルサイクラーにセットし、95℃で2分、95℃で30秒、60℃で30秒、72℃で2分、30サイクル、72℃で10分の条件で反応を行い、ErbB1細胞外ドメイン(ECD)遺伝子を増幅した。ErbB1のN末端側にXhoIサイトを作り、ErbB1の膜貫通ドメインの上流にAgeIサイトを作った。反応液をエタチンメイト(NIPPON GENE)を使ってエタノール沈澱させ沈澱を18μlの滅菌UPWに溶解し、そこに10×PCRバッファ(SIGMA)を2.5μl、10mM dNTP(SIGMA)を4μl、Taq DNA polymerase(SIGMA)を0.5μl加えて72℃で1時間反応させ、先に増幅したErbB1 ECD遺伝子の3'末端にアデニンをテイリングさせた。この反応液をアガロース電気泳動して、2kbpのバンドを切り出してそこから、CONCERTTM Rapid Gel Extraction System(LIFE TECHNOLOGIES)でDNAを抽出した。そしてこれをエタノール沈殿し、沈殿を10μlの滅菌UPWに溶解した。アガロース電気泳動でErbB1細胞外ドメイン(ErbB1 ECD)のバンドを確認し、ErbB1細胞外ドメインとpCR2.1(TA cloning vector, Invitrogen)のモル比を100:1で混合しT4 DNA リガーゼ(NIPPON GENE)を1μl加え、16℃水槽で一晩反応させた。このライゲーション反応液の全量を無菌的にコンピテントセルXL-1 Blueに加え、30分間氷上に置き、42℃で1分間ヒートショックして、即氷上に置き充分冷却してからSOC培地を400μl加えて37℃で1時間培養して形質転換を行った。培養液を50μlの20mg/ml X-Galと10μlの100mM イソプロピルチオガラクトシド(IPTG)とともにLB-Amp50 Agarに塗り広げ37℃で一晩培養して青白選択を行った。出てきたコロニーの内、白コロニーを採り100μlのLB-Amp50に植菌して7時間、280/分で振盪培養した。これを3分間遠心して上清を吸引し、ペレットを50μlの1% Triton X-100で溶菌した。そしてこの液を1μl、10μMフォワードプライマー♯547(配列番号27)、10μMリバースプライマー♯548(配列番号8)をそれぞれ1μl、2.5mM dNTPを2μl、10×PCRバッファを2.5μl、滅菌UPWを17.25μl、Taq DNA ポリメラーゼを0.25μlを混合しサーマルサイクラーにセットし、95℃で10分、94℃で1分、50℃で1分、72℃で1分、30サイクル、72℃で10分の条件で反応させた。反応液をアガロース電気泳動し、700bpのバンドがみえるもの3クローンを5mlのLB-Amp50に植菌して、37℃で一晩振盪培養した。そして、QIAprep Spin Miniprep Kitでプラスミドを抽出して、XhoI、AgeIの消化およびEcoRIによりクローニングベクターへのErbB1 ECD挿入を確認した。更にDNAシーケンサでErbB1 ECDの全塩基配列を確認した(用いたプライマーは♯361(配列番号7)、♯547(配列番号27)、♯548(配列番号8)、M13(-21)primer(配列番号14)(Applied Biosystems))。配列を確認したプラスミドはXhoI、AgeIで切断し反応液をアガロース電気泳動してErbB1 ECD(2kbp)のバンドを切り出した。切り出したゲルからCONCERTTM Rapid Gel Extraction SystemでDNAを抽出し、更にエタチンメイトでエタノール沈澱し、沈澱を10μlの滅菌UPWに溶解した。同時に、pEGFP-N1(pBO315)をXhoIとAgeIで切断し、70℃で15分おいて制限酵素を失活させた。そして、ErbB1 ECD遺伝子とpBO315のモル比が100:1となるように混合しT4 DNAリガーゼ(NEB)を1μl加えて、16℃で一晩反応させた。反応液を全量XL-1 Blueに導入して形質転換し50μg/mlカナマイシン含有 LB寒天培地(LB-Kn50 Agar)に植菌して37℃で一晩培養した。出現コロニーを5個取り50μg/mlカナマイシン含有 LB培地(LB-Kn50)5mlにそれぞれ植菌し一晩振盪培養した。QIAprep Spin Miniprep Kitでプラスミドをそれぞれ抽出しXhoIおよびAgeIで切断しpBO315にErbB1 ECDが挿入されたことを確認した。確認後、1つのクローンをAgeI、NotIで切断しpBO315からEGFPの配列を排除した。アガロース電気泳動を行い6kbpの断片を切り出した。この切り出したゲルからCONCERTTM Rapid Gel Extraction SystemでDNAを抽出し10μlの滅菌UPWに溶解させた。
human fetal heart cDNAを1μl、10×pfxバッファを5μl、10×エンハンサーを5μl、50mM Mg2SO4を1μl、10μM フォワードプライマー♯532(配列番号3)、10μMリバースプライマー♯533(配列番号4)をそれぞれ1μlずつ、2.5mM dNTPを6μl、滅菌UPWを29μl、PLATINUM pfx DNAポリメラーゼ(GIBCO BRL)を1μlを0.2mlチューブに混合しサーマルサイクラーにセットして、94℃で2分、94℃で1分、55℃で1分、68℃で5分、35サイクル、68℃で10分の条件で反応させて、ErbB4遺伝子の全長を増幅させた。続いてこの反応液を1μl、10×PFUバッファを5μl、10μMフォワードプライマー♯534(配列番号5)、10μMリバースプライマー♯535(配列番号6)をそれぞれ1μlずつ、2.5mM dNTPを6μl、滅菌UPWを35.5μl、PFU turbo DNA ポリメラーゼを0.5μlを0.2mlチューブに加えて混ぜ、サーマルサイクラーにセットして、95℃で10分、95℃で30秒、65℃で30秒、72℃で2分30秒、35サイクル、72℃で10分の条件で反応させて、ErbB4細胞外ドメイン(ECD)遺伝子を増幅しN末端側にBglIIサイトを、そしてErbB4 膜貫通ドメイン上流にはAgeIサイトを作った。反応液をエタチンメイトを使って18μlの滅菌UPWに溶解した。これに、10×PCRバッファを2.5μl、10mM dATPを3μl、2.5mM dNTPを1μl、Taq DNAポリメラーゼは0.5μlそれぞれ加えて、72℃で1時間反応させて、DNAの3'末端にアデニンをテイリングさせた。また、上記のErbB1の操作と同様にしてTAクロ−ニングベクターのpCR2.1とライゲーションさせ、XL-1 Blueに形質転換し植菌する時に、X-Gal、IPTGを同時に加えて培養し青白選択を行った。白コロニーをErbB4 ECDの660bpを増幅するプライマー♯549、♯550(配列番号9、10)を用いてダイレクトPCRを行い、TAクロ−ニングベクターへのErbB4 ECDの挿入を確認した。更に、制限酵素AgeI、BglIIの消化および、EcoRIによる切断で挿入を確認した。また、プライマー♯361(配列番号7)、♯549(配列番号9)、♯550(配列番号10)、♯563(配列番号11)、♯564(配列番号13)、M13(-21)プライマー(配列番号14)を用いて、DNA塩基配列を確認した。確認後、ベクターをAgeI、BglIIで切断しアガロース電気泳動をしてErbB4 ECDのバンドを切り出した。ゲルからCONCERTTM Rapid Gel Extraction SystemでDNAを抽出し10μlの滅菌UPWに溶解させた。同時にpBO315もAgeI、BglIIで切断し反応液を65℃で15分置いて、制限酵素を失活させた。そしてErbB4 ECD遺伝子とFLAG-tagのモル比が1:100になるように混合し、T4 DNAリガーゼを1μl加え、16℃で一晩反応させた。そして、反応液の全量をXL-1 Blueの形質転換に用い形質転換体をLB-Kn50 Agarに植菌して、37℃で一晩培養した。出現コロニーを5mlのLB-Kn50に植菌し一晩培養してQIAprep Spin Miniprep Kitでプラスミドを抽出し、AgeI、BglIIで切断してスクリーニングした。以下、1-1と同様にして、FLAG-tagを挿入し、そして、プラスミドを塩化セシウム密度勾配超遠心により大量精製した。
まず、ErbB1 ECD-FLAGベクター、ErbB4 ECD-FLAGベクター500ngをAgeIで切断した。反応液をエタチンメイトでエタノール沈澱し、沈澱を乾燥させてから17μlの滅菌UPWに溶かした。これに2μlのBacterial Alkaline Phosphatase(BAP)バッファ(TaKaRa)を加え、更にBAP(TaKaRa)を1μl加えて65℃で1時間反応させた。反応後滅菌UPWを80μl加え、更にフェノール/クロロホルムイソアミルアルコール(CIAA, クロロホルム:イソアミルアルコール=24:1)を100μl加えボルテックスした。室温で15分間遠心し水層(上層、無色透明)をピペットマンで回収し新しいチューブに移した。そこへ、再びフェノール/CIAAを加え同様の操作を2回繰り返した。最後に、CIAAを100μl加えボルテックスし、5分間遠心して水槽を回収した。そして、3M NaOAcを5μl加えエタノールを100μl加えて良く混ぜドライアイスエタノールバスに10分間置いた後10分間遠心して上清を除去し70%エタノールで沈澱を洗い、それから沈澱を乾かして10μlの滅菌UPWに溶解した。
2-1. ErbB ECD-ヒンジ-FLAG発現HEK293H細胞の樹立
ヒト胚性腎293H(HEK293H)細胞を293 SFM II(GIBCO)で培養した。細胞を回収し8×106個ずつ2本の1.5mLチューブに分注した。これを遠心して上清を除去しEP 培地(10mM グルコース、100μM DTT/RPMI1640)を800μLずつ加えて懸濁した。そしてプラスミドDNA:ErbB1 ECD-ヒンジ-FLAG発現ベクター(pBO547)またはErbB4 ECD-ヒンジ-FLAG発現ベクター(pBO548)を10μgずつ加え4mmギャップエレクトロポレーションキュベット(MβP)に移した。そしてGene pulser II(BIO-RAD)を用い0.26kV-950μFでプラスミドを導入した。T75cm2細胞培養ボトルに10% FCS-DMEMを入れ、エレクトロポレーションした細胞を加えた。そして、37℃、5% CO2で24時間培養した。それから、培養液を回収し1200rpm、5分、4℃で遠心し上清を除去し細胞を2mLずつの1mg/mL G418、100μg/mL カナマイシン / 293 SFM II(1mg/mL G418-293 SFM II)に懸濁し、生細胞数を計数した(ErbB1 ECD-ヒンジ-FLAG:8.2×105個、ErbB4 ECD-ヒンジ-FLAG:6.4×105個)。そしてT75cm2ボトルに18mLの1mg/mL G418-293 SFM IIを入れ、細胞を加えた。17〜21日間培養してG418耐性の細胞を選択した。
175cm2細胞培養用フラスコ(BD FalconTM)に60mLの1mg/mL G418、100μg/mL カナマイシン入り293 SFM-II(GIBCO)を入れ3.5×106個のHEK293H/ ErbB ECD-ヒンジ-FLAG発現ベクターpBO547またはpBO548を撒いて37℃、5% CO2で6〜8日間培養した。この培養液を600〜800mL回収し、5,000rpm、10min、4℃で遠心(HITACHI himac CR20, rotor NO. 30)し上清を精製に用いた。TALON 2mL Disporsable Gravity Column(CLONTECH)にリン酸緩衝化食塩水(PBS)、pH7.4を2回通して濯いだ。それからAnti-FLAG(登録商標)M2 Agarose Affinity Gel(SIGMA)1mLをカラムに充填した。次にPBSをゲルのグリセリンが抜けるまで流し、そして、0.1M リン酸バッファ(pH3.5)を1mLずつ3回流してゲルを洗った。続いてPBSを1mLずつ5回カラムに通しカラムを平衡化した。次に培養上清をカラムに通した。その後、20mLのPBSでカラムを洗い、0.1M リン酸バッファ(pH3.5)でレセプターを溶出した。溶出液は1mLに対して2M リン酸バッファ(pH8.0)を90μLで受けて中和した。
ビオチン-(AC5)2-スルホ-OSu(スルホスクシンイミジルN-[N'-(D-ビオチニル)-6-アミノヘキサノイル]-6'-アミノヘキサノエート)(同仁化学)をUPWに溶解して3.3mMの溶液を作った。モル比でErbB ECD-ヒンジ-FLAG:ビオチン=1:12となるように混合し室温で4時間反応させた。反応を0.1Mグリシン50μLを加えて止めて、反応液の全量をUPWで2.5mLにして、PBSで平衡化したPD-10カラム(Amersham pharmacia biotech)にアプライして3mLのPBSで溶出してバッファ交換した。
(実施例3)リガンドの調製
rhBTCの精製は、Seno et al.(1996)Human Betacellulin, a Member of the EGF Family Dominantly Expressed in Pancreas and Small Intestine, is Fully Active in a Monomeric Form.Growth Factors 13, 181-191に従って行った。
Myc-BTC-HA発現プラスミド(pBO651)を構築し、E.coli BL21(DE3)pLysSに導入し、50μg/mLアンピシリン、10μg/mLクロラムフェニコール含有LB培地でOD660が0.3になるまで培養した。その後、イソプロピルチオガラクトシド(IPTG)を終濃度0.4mMとなるように加え、更に3時間培養した。培養液を遠心して菌体を回収し、-80℃で凍結し、37℃で解凍した。菌体ペレットを培養液の1/50量の50mMリン酸バッファ(pH7.4)で懸濁し氷上に1時間おいて溶菌し、更にソニケーションした。遠心して上清を回収しMyc-BTC-HA粗抽出物とした。
25mm(幅)x75mm(長さ)x1mm(厚み)のスライドガラスを、ポリアリルアミン水溶液(0.1g/l)に浸漬することにより、静電層を形成した。その後、静電層のアミノ基に、多価カルボン酸としてのポリアクリル酸を、0.1Mの1-[3-(ジメチルアミノ)プロピル]-3-エチルカルボジイミドの存在下で縮合した。そして、0.1M リン酸緩衝液(pH6)300mlに0.1mの1-[3-(ジメチルアミノ)プロピル]-3-エチルカルボジイミド・塩酸塩と20mMのN-ヒドロキシスクシンイミドを溶解した活性化液中に30分間浸漬することによって活性化した。
96穴プレートにrhBTCを0.2mg/mLから2倍の系列希釈で、Myc-BTC-HAを含む細胞粗抽出液または対照としてMyc-BTC-HAを発現しないベクター(NC)を含む細胞粗抽出液を1.2倍と2倍から2倍の系列希釈でサンプルを調製した。各サンプルは10%グリセリンを含む。活性化した実施例4の固体支持体にGT-MASS SYSTEM(日本レーザー電子)でサンプルをスポットし、飽和食塩水を含ませた濾紙を敷いたシャーレに入れて37℃で1時間反応させた。次に15mLの2% BSA、0.1M Tris-Cl(pH7.4)/PBSに固体支持体を浸けて室温で1時間ブロッキングを行った。0.1% Tween-20含有PBS(PBST)で3回固体支持体を洗い遠心した、その後、1% BSAを含む100μg/mLビオチン化ErbB1 ECD-ヒンジ-FLAGを30μLかけてカバーグラスで覆い、室温で1時間反応させた。PBSTで3回洗い遠心した。そして、0.1% BSAを含むPBSに10μg/mL アビジン-Cy3、1μg/mL BSA-ビオチンを混合した液(反応30分前に調製)を30μLかけてカバーグラスで覆い室温で1時間反応させた。PBSTで4回洗い最後にPBSで1回濯いで遠心し、GTMASS SCANNERでCy3の蛍光を読み取った。蛍光画像を図3に示す。この蛍光画像においては、右方向へ同一の試料を連続してスポットしているので水平に並ぶ3点については組み換えベータセルリンの濃度がほぼ一定、上方向に向けて試料を2倍ずつ希釈したものをスポットしているので組み換えベータセルリンの濃度は上へ行く程低くなっている。蛍光強度をGTMASS ANALYSISを用いて解析した結果を図4に示す。以上の結果から、ErbBと相互作用するポリペプチドの固定化量が多いスポットほど、ErbB細胞外ドメインが多く結合していることがわかる。以上から、本発明の方法により、ポリペプチドと膜レセプターとの相互作用を検出できることが示された。
Claims (7)
- 担体上にポリペプチドを固定化し、これに細胞表面上の膜レセプターにおける細胞外ドメインを含むが膜結合に必須の領域を含まないレセプター誘導体を反応させ、固定化したポリペプチドとレセプター誘導体との相互作用を検出することにより、ポリペプチドとレセプターとの相互作用を検出する方法。
- 担体が、基板上にポリペプチドと共有結合しうる官能基、ならびにダイヤモンド、軟ダイヤモンド、炭素系物質および炭化物から選ばれる少なくとも1種の表面層を有する固体支持体である、請求項1記載の方法。
- ポリペプチドおよび多価アルコールを含むスポッティング溶液を担体上にスポッティングすることによりポリペプチドを担体上に固定化する請求項1または2記載の方法。
- 担体上に固定化するポリペプチドが、膜レセプターに対するリガンドまたはリガンド変異体である請求項1〜3のいずれか1項記載の方法。
- 請求項4記載の方法を用いて、膜レセプターに対するリガンドの変異体をスクリーニングする方法。
- 請求項1〜3のいずれか1項記載の方法を用いて、膜レセプターに対するリガンドをスクリーニングする方法。
- 請求項1〜4のいずれか1項記載の方法を用いる診断方法。
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JP2007282591A (ja) * | 2006-04-19 | 2007-11-01 | Yamaguchi Univ | スタスミンチップ及びこれを用いたスタスミン結合タンパク質の検出方法 |
JP2008105973A (ja) * | 2006-10-24 | 2008-05-08 | Toyo Kohan Co Ltd | ポリペプチド固定化担体の保存方法 |
JP2010539497A (ja) * | 2007-09-18 | 2010-12-16 | エーアーデーエス・ドイッチェランド・ゲゼルシャフト ミット ベシュレンクテル ハフツング | バイオセンサを再生する装置及び方法 |
JP2011122957A (ja) * | 2009-12-11 | 2011-06-23 | Tosoh Corp | 高特異的かつ高感度なタンパク質検出方法 |
JP2018529950A (ja) * | 2015-09-02 | 2018-10-11 | ベンタナ メディカル システムズ, インコーポレイテッド | 解析的に別個の検体染色のパターンの混合を有する細胞サンプルの自動化解析 |
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WO2002018444A2 (en) * | 2000-09-01 | 2002-03-07 | Genentech, Inc. | Erbb4 antagonists |
WO2002044727A1 (fr) * | 2000-11-30 | 2002-06-06 | Chugai Seiyaku Kabushiki Kaisha | Procede de mesure de l'activite de liaison d'une proteine se liant a un ligand presentant une faible stabilite chimique a un premier ligand |
WO2003031620A1 (fr) * | 2001-10-02 | 2003-04-17 | Mochida Pharmaceutical Co., Ltd. | Nouveau recepteur des cytokines de classe ii |
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WO2002018444A2 (en) * | 2000-09-01 | 2002-03-07 | Genentech, Inc. | Erbb4 antagonists |
WO2002044727A1 (fr) * | 2000-11-30 | 2002-06-06 | Chugai Seiyaku Kabushiki Kaisha | Procede de mesure de l'activite de liaison d'une proteine se liant a un ligand presentant une faible stabilite chimique a un premier ligand |
WO2003031620A1 (fr) * | 2001-10-02 | 2003-04-17 | Mochida Pharmaceutical Co., Ltd. | Nouveau recepteur des cytokines de classe ii |
Cited By (6)
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JP2007282591A (ja) * | 2006-04-19 | 2007-11-01 | Yamaguchi Univ | スタスミンチップ及びこれを用いたスタスミン結合タンパク質の検出方法 |
JP2008105973A (ja) * | 2006-10-24 | 2008-05-08 | Toyo Kohan Co Ltd | ポリペプチド固定化担体の保存方法 |
JP2010539497A (ja) * | 2007-09-18 | 2010-12-16 | エーアーデーエス・ドイッチェランド・ゲゼルシャフト ミット ベシュレンクテル ハフツング | バイオセンサを再生する装置及び方法 |
US9162259B2 (en) | 2007-09-18 | 2015-10-20 | Eads Deutschland Gmbh | Device and method for the regeneration of biosensors |
JP2011122957A (ja) * | 2009-12-11 | 2011-06-23 | Tosoh Corp | 高特異的かつ高感度なタンパク質検出方法 |
JP2018529950A (ja) * | 2015-09-02 | 2018-10-11 | ベンタナ メディカル システムズ, インコーポレイテッド | 解析的に別個の検体染色のパターンの混合を有する細胞サンプルの自動化解析 |
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