JP2012531396A - 診断方法のためのigfbp−4断片の検出 - Google Patents
診断方法のためのigfbp−4断片の検出 Download PDFInfo
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Abstract
【選択図】なし
Description
本発明においては、IGFBP−4のN末端タンパク質分解断片およびC末端タンパク質分解断片を、ACS患者や数種の癌患者の血漿試料において、健常ドナーの血漿よりも有意に高いレベルで存在するバイオマーカーとして開示する。IGFBP−4断片の免疫アッセイは、ACSまたは癌の早期発見、あるいは疾患発症の危険度を決定するために使用することができる。
マウスの免疫のために入手した合成ペプチド:
ペプチド−1(配列番号1)
ペプチド−2(配列番号2)
1群が5匹のBALB/cマウスを、ペプチド−タンパク質結合体で5回免疫した。
グループ1.1次免疫:10μgのBSA−ペプチド−1を含有するPBS溶液0.2mlを60%のフロインド完全アジュバントと共に腹腔内投与。2次免疫:30日目に、5μgのBSA−ペプチド−1を含有するPBS溶液0.2mlを60%のフロインド完全アジュバントと共に腹腔内投与。3次免疫:60日目に、2.5μgのBSA−ペプチド−1を含有するPBS溶液0.2mlを腹腔内投与。
選択したハイブリドーマクローンを腹腔内に注射し、モノクローナル抗体をマウス腹水中で産生した。タンパク質Aアフィニティークロマトグラフィーを用いて、腹水から抗体を精製した。樹脂はGE Healthcare Life Sciences(ニュージャージー州、ピスカタウェイ)より入手し、精製は製造者の説明書に従って実施した。精製モノクローナル抗体は、50%硫酸アンモニウムの懸濁液として4℃で保存した。
選択したモノクローナル抗体の特異性を確認するために、IGFBP−4タンパク質分解断片を得た。PAPP−A依存性タンパク質分解反応を、既報(14)の条件に従って実施した。2μgのヒト組み換えIGFBP−4を、2mMのCaCl2、1.8μgのIGF−II(ミズーリ州、セントルイス、Sigma Chemicalsより入手)、40ngのヒト組み換えPAPP−A(フィンランド国、トゥルク、HyTest製)および2μlのタンパク質分解酵素阻害剤カクテル(ミズーリ州、セントルイス、Sigma Chemicalsより入手)の存在下で、0.23mlの50mM Tris−HCl(pH7.5)中でインキュベートした。反応は37℃で15時間実施し、試料を−20℃で凍結することによって反応を停止した。IGFBP−4のPAPP−A依存性開裂の程度は、Abcam社(マサチューセッツ州、ケンブリッジ)より入手した特異的ウサギポリクローナル抗体 1μg/mlを用いたウェスタンブロッティングで決定した(図1)。
アフィニティー精製したモノクローナル抗体の特異性を、サンドイッチ免疫アッセイでも確認した(図2B)。IGFBP−4のタンパク質分解断片(N末端断片またはC末端断片)に特異的な1種のモノクローナル抗体(間接ELISAにおける全長分子との交差反応性は5%未満)、および完全なIGFBP−4いずれかのエピトープを認識する別のMAbを用いる、数種のサンドイッチアッセイを開発した。完全なIGFBP−4に特異的なマウスモノクローナル抗体の作製については、実施例2に記載した。
マウスの免疫
5匹のBALB/cマウスを、哺乳動物NS0細胞系で発現させたヒト組み換えIGFBP−4で5回免疫した。このタンパク質は、ミズーリ州、セントルイス、Sigma Chemicalsより入手した。1次免疫:5μgのIGFBP−4を含有するPBS溶液0.2mlを60%のフロインド完全アジュバントと共に腹腔内投与。2次免疫:30日目に、2μgのIGFBP−4を含有するPBS溶液0.2mlを60%のフロインド完全アジュバントと共に腹腔内投与。3次免疫:60日目に、2μgのIGFBP−4を含有するPBS溶液0.2mlを腹腔内投与。
選択したハイブリドーマクローンを腹腔内注射し、全長IGFBP−4に特異的なモノクローナル抗体をマウス腹水中で産生した。タンパク質Aアフィニティークロマトグラフィーを用いて、腹水から抗体を精製した。樹脂はGE Healthcare Life Sciences(ニュージャージー州、ピスカタウェイ)より入手し、精製は製造者の説明書に従って実施した。精製モノクローナル抗体は、50%硫酸アンモニウムの懸濁液として4℃で保存した。
ヒト アテローム性動脈硬化症の冠状血管試料は、使用するまで−70℃で保存した。アテローム性動脈硬化症の冠状動脈からのPAPP−Aの抽出は、0.15MのNaCl、0.5%のTritonX100およびタンパク質分解酵素阻害剤カクテルを含有する50mMのTris−HCl(pH7.8)緩衝液中で組織をホモジェナイズした後に行った。抽出したPAPP−Aは、アフィニティークロマトグラフィーによって精製した。PAPP−Aの精製に用いたアフィニティーマトリックスは、PAPP−A特異的モノクローナル抗体4G11(フィンランド国、トゥルク、HyTestより入手)を用いて調製した。精製タンパク質がPAPP−Aであることを確認するために、数種のPAPP−A特異的モノクローナル抗体を用いたウェスタンブロッティングおよび液体クロマトグラフィー/タンデム質量分析法を使用した。
(ST上昇型の)ACS患者43人の血液と、健常ドナー由来の34例の血漿試料について、断片特異的なサンドイッチ免疫アッセイで試験した。全ての血漿試料をEDTAの存在下で採取し、測定まで−70℃で保存した。
Claims (24)
- IGFBP−4の新規エピトープを認識する抗体であって、該エピトープは、配列番号3に記載のアミノ酸配列または配列番号3と少なくとも80%の相同性を有するアミノ酸配列を包含するIGFBP−4の酵素依存性開裂によって発生するものであることを特徴とする抗体。
- IGFBP−4の酵素依存性開裂によって発生する新規エピトープを、全長IGFBP−4を認識するよりも高い親和性をもって特異的に認識することを特徴とする、請求項1に記載の抗体。
- IGFBP−4の新規エピトープを特異的に認識し、且つ、全長IGFBP−4を認識しない抗体であって、該エピトープは、配列番号3に記載のアミノ酸配列または配列番号3と少なくとも80%の相同性を有するアミノ酸配列を包含するIGFBP−4の酵素依存性開裂によって発生するものであることを特徴とする抗体。
- IGFBP−4のM135とK136との間で生じる酵素依存性開裂によって発生する新規エピトープを特異的に認識し、且つ、全長IGFBP−4を認識しないことを特徴とする、請求項3に記載の抗体。
- IGFBP−4のM135とK136との間で生じる酵素依存性開裂によって発生する新規エピトープを特異的に認識することを特徴とする、請求項1に記載の抗体。
- IGFBP−4のM135とK136との間で生じる酵素依存性開裂によって発生する新規エピトープを、全長IGFBP−4を認識するよりも高い親和性をもって特異的に認識することを特徴とする、請求項5に記載の抗体。
- IGFBP−4のM135とK136との間で生じるPAPP−Aによる開裂の結果として発生する新規エピトープを、全長IGFBP−4を認識するよりも高い親和性をもって特異的に認識することを特徴とする、請求項1に記載の抗体。
- 請求項1〜4のいずれかの抗体と同等の特異性を有するアプタマーまたはその他のバインダー。
- モノクローナル抗体またはその断片である、請求項1〜4のいずれかに記載の抗体。
- ポリクローナル抗体またはその断片である、請求項1〜4のいずれかに記載の抗体。
- 組み換え抗体またはその断片である、請求項1〜4のいずれかに記載の抗体。
- 請求項1〜4の抗体から選ばれる少なくとも1種を用いる、IGFBP−4のタンパク質分解断片を定性的または定量的に検出する方法。
- 請求項8のアプタマーまたはその他のバインダーから選ばれる少なくとも1種を用いる、IGFBP−4のタンパク質分解断片を定性的または定量的に検出する方法。
- 診断的免疫アッセイ法であることを特徴とする、請求項12または13に記載の方法。
- 循環器系疾患の診断用である、請求項14に記載の方法。
- 癌の診断用である、請求項14に記載の方法。
- 内因性IGFBP−4タンパク質分解断片、組み換えIGFBP−4タンパク質分解断片、またはIGFBP−4の酵素依存性開裂によって発生する新規エピトープに相当するエピトープを包含する合成ペプチドを標準物質として用いて検量曲線を作製することを包含し、診断方法として使用することを特徴とする、請求項12または13に記載の方法。
- 検出されたIGFBP−4タンパク質分解断片のレベルを、作製した検量曲線と比較することを更に包含する、請求項17に記載の方法。
- 免疫アッセイ法が、第1の捕捉抗体および第2の検出抗体を用いるサンドイッチ免疫アッセイ法である、請求項14に記載の方法。
- 請求項1〜4のいずれかのモノクローナル抗体および請求項8のアプタマーまたはバインダーからなる群より選ばれる少なくとも1種を包含する免疫アッセイにおける、内因性IGFBP−4タンパク質分解断片、組み換えIGFBP−4タンパク質分解断片、またはIGFBP−4の酵素依存性開裂によって発生する新規エピトープに相当するエピトープを包含する合成ペプチドの、標準物質または検量物質としての使用。
- 請求項1〜4のいずれかの抗体と同等の特異性を有する抗体の製造における、内因性IGFBP−4タンパク質分解断片、組み換えIGFBP−4タンパク質分解断片、またはIGFBP−4の酵素依存性開裂によって発生する新規エピトープに相当するエピトープを包含する合成ペプチドの、抗原としての使用。
- 患者試料中のIGFBP−4タンパク質分解断片の診断的アッセイを行うための免疫アッセイキットであって、
(a)請求項1〜4のいずれかの抗体と同等の特異性を有する抗体、
(b)全長IGFBP−4のいずれかのエピトープに特異的な他の抗体、および
(c)請求項20において定義した標準物質または検量物質
を包含するキット。 - 請求項1〜4のいずれかのモノクローナル抗体および請求項8のアプタマーまたはバインダーからなる群より選ばれる少なくとも1種を用いるアッセイの使用によって、患者試料中のPAPP−Aのタンパク質分解活性を測定することを包含する診断方法。
- 請求項23の診断方法のための標準物質または検量物質である、酵素学的に活性な内因性PAPP−Aまたは組み換えPAPP−A。
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US22122509P | 2009-06-29 | 2009-06-29 | |
FI20095733A FI20095733A0 (fi) | 2009-06-29 | 2009-06-29 | IGFBP-4-fragmenttien määrittäminen diagnostisena menetelmänä |
US61/221,225 | 2009-06-29 | ||
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JP2015518964A (ja) * | 2012-06-06 | 2015-07-06 | アトークアント・ダイアグノスティクス・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツングAttoquant Diagnostics Gmbh | 血液試料中のタンパク質分解カスケードのペプチド分解産物の測定方法 |
JP2018193372A (ja) * | 2018-05-29 | 2018-12-06 | 株式会社森永生科学研究所 | 抗ペプチド抗体の製造方法及び設計方法 |
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FI20115367A0 (fi) * | 2011-04-15 | 2011-04-15 | Hytest Oy | Menetelmä sydän- ja verisuonitapahtumien määrittämiseksi käyttämällä IGFBP-fragmentteja |
GB201121924D0 (en) * | 2011-12-20 | 2012-02-01 | Fahy Gurteen Labs Ltd | Detection of breast cancer |
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JP2018193372A (ja) * | 2018-05-29 | 2018-12-06 | 株式会社森永生科学研究所 | 抗ペプチド抗体の製造方法及び設計方法 |
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CN102471379B (zh) | 2019-08-06 |
FI20095733A0 (fi) | 2009-06-29 |
US9012610B2 (en) | 2015-04-21 |
EP2448969B1 (en) | 2015-03-04 |
US9964549B2 (en) | 2018-05-08 |
EP2448969A4 (en) | 2013-01-23 |
EP2448969A1 (en) | 2012-05-09 |
WO2011001029A1 (en) | 2011-01-06 |
JP5840605B2 (ja) | 2016-01-06 |
US20150219671A1 (en) | 2015-08-06 |
KR20120102499A (ko) | 2012-09-18 |
CN102471379A (zh) | 2012-05-23 |
KR101767611B1 (ko) | 2017-08-23 |
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