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Generation of recombinant antibody libraries that facilitate highly sensitive monitoring of low molecular weight biomarkers

Research Project

Project/Area Number 22590542
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Laboratory medicine
Research InstitutionKobe Pharmaceutical University

Principal Investigator

KOBAYASHI Norihiro  神戸薬科大学, 薬学部, 教授 (90205477)

Co-Investigator(Kenkyū-buntansha) OYAMA Hiroyuki  神戸薬科大学, 薬学部, 助教 (80572966)
Co-Investigator(Renkei-kenkyūsha) KOYAMA Junko  神戸薬科大学, 薬学部, 講師 (60102109)
MORITA Izumi  神戸薬科大学, 薬学部, 助手 (20299085)
Project Period (FY) 2010 – 2012
Project Status Completed (Fiscal Year 2012)
Budget Amount *help
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2012: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2011: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2010: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Keywordsバイオマーカー / モニタリング / 抗体 / 抗体ライブラリー / 抗体工学 / ライブラリー / エストラジオール / ジゴキシン
Research Abstract

To generate high-performance scFv mutants that are specific to small biomarkers, we constructed a hapten- targeting scFv library based on a single master-frame of which VH and VL subgroups are those frequently used in anti-hapten antibodies. However, this library failed to yield any practical binder, suggesting that repeated trials are necessary to select suitable positions/patterns for mutagenesis. Thus, we decided to use streptavidin (stav) as a new scaffold, and generated a library of stav mutatants, each member of which has random mutations in their loop structures. After several rounds of selection, wefound mutant clones that gained binding abilities to estradiol or cortisol.

Report

(4 results)
  • 2012 Annual Research Report   Final Research Report ( PDF )
  • 2011 Annual Research Report
  • 2010 Annual Research Report
  • Research Products

    (38 results)

All 2013 2012 2011 2010

All Journal Article (9 results) (of which Peer Reviewed: 9 results) Presentation (22 results) Book (6 results) Patent(Industrial Property Rights) (1 results)

  • [Journal Article] Toward in vivo imaging of heart disease using a radiolabeled single-chain Fv fragment targeting tenascin-C2011

    • Author(s)
      Kobayashi N, Odaka K, Uehara T, Imanaka-Yoshida K, Kato Y, Oyama H, Tadokoro H, Akizawa H, Tanada S, Hiroe M, Fukumura T, Komuro I, Arano Y, Yoshida T, Irie T
    • Journal Title

      ANALYTICAL CHEMISTRY

      Volume: 83 Issue: 23 Pages: 9123-9130

    • DOI

      10.1021/ac202159p

    • Related Report
      2012 Final Research Report 2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] A monoclonal antibody-basedenzyme-linked immunosorbent assay for human urinary cotinine to monitor tobacco smoke exposure.2011

    • Author(s)
      Kobayashi N., Banzono E., Shimoda Y.,Oyama H., Kunihiro T., Morita I., Ohta M.
    • Journal Title

      Anal. Methods

      Volume: 82 Issue: 3 Pages: 1027-1038

    • DOI

      10.1021/ac902283n

    • Related Report
      2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] Antibody engineering toward high-sensitivity high-throughput immunosensing of small molecules.2011

    • Author(s)
      Kobayashi N., Oyama H.
    • Journal Title

      Analyst

      Volume: 136(4) Issue: 4 Pages: 642-651

    • DOI

      10.1039/c0an00603c

    • Related Report
      2012 Final Research Report 2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] A monoclonal antibody-based enzyme-linked immunosorbent assay for human urinary cotinine to monitor tobacco smoke exposure2011

    • Author(s)
      Kobayashi N., Banzono E., Shimoda Y., Oyama H., Kunihiro T., Morita I., Ohta M.
    • Journal Title

      Anal.Methods

      Volume: 3(9) Pages: 1995-2002

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Oligosaccharide-assisted direct immunosensing of small molecules.2010

    • Author(s)
      Kobayashi N., Oyama H., Suzuki I., Kato Y.,Umemura T., Goto J.
    • Journal Title

      Anal.Chem.

      Pages: 4333-4336

    • URL

      http://pubs.acs.org/doi/full/10.1021/ac100865p

    • Related Report
      2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] LC-MS/MS coupled with immunoaffinity extraction for determination of estrone,17ss-estradiol and estrone 3-sulfate in human plasma.2010

    • Author(s)
      Hosogi J., Tanaka H., Fujita K., Kuwabara T., Ikegawa S., Kobayashi N., Mano N., Goto J.
    • Journal Title

      J. Choromatogr. B

      Volume: 878 Issue: 2 Pages: 222-227

    • DOI

      10.1016/j.jchromb.2009.08.010

    • Related Report
      2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] Two-step in vitro antibody affinity maturation enables estradiol-17β assays with more than 10-fold higher sensitivity.2010

    • Author(s)
      Kobayashi N.; Oyama H.; Kato Y.; Goto J.; Soderlind E.; Borrebaeck A.K.C.
    • Journal Title

      Anal.Chem.

      Volume: 82 Pages: 1027-1038

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Oligosaccharide-assisted direct immunosensing of small molecules.2010

    • Author(s)
      Kobayashi N.; Oyama H.; Suzuki I.; Kato Y.; Umemura T.; Goto J.
    • Journal Title

      Anal.Chem.

      Volume: 82 Pages: 4333-4336

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Journal Article] LC-MS/MS coupled with immunoaffinity extraction for determination of estrone, 17β-estradiol and estrone 3-sulfate in human plasma.2010

    • Author(s)
      Hosogi J.; Tanaka H.; Fujita K.; Kuwabara T.; Ikegawa S.; Kobayashi N.; Mano N.; Goto J.
    • Journal Title

      J.Choromatogr.B

      Volume: 878 Pages: 222-227

    • NAID

      10028031536

    • Related Report
      2010 Annual Research Report
    • Peer Reviewed
  • [Presentation] ハプテンの高感度測定を目的とする抗イディオタイプ抗体scFv-酵素融合体の調製と諸性質2013

    • Author(s)
      大山浩之,小林典裕,加藤芳徳,丹羽俊文
    • Organizer
      日本薬学会第130年会
    • Place of Presentation
      岡山
    • Year and Date
      2013-03-29
    • Related Report
      2012 Final Research Report
  • [Presentation] 実用診断試薬を創出する試験管内分子進化 (5). 改良型scFvを用いるヒト血清中エストラジオールの測定2013

    • Author(s)
      大山 浩之,中田 茂利,丹羽 俊文,小林 典裕
    • Organizer
      日本薬学会第132年会
    • Place of Presentation
      横浜
    • Related Report
      2012 Annual Research Report
  • [Presentation] 実用診断試薬を創出する試験管内分子進化(6).改良型scFvを用いるヒト尿中コチニンELISAの確立2013

    • Author(s)
      森田 いずみ,大山 浩之,渡部 芳郎,平井 杏奈,太田 光熙,小林 典裕
    • Organizer
      日本薬学会第132年会
    • Place of Presentation
      横浜
    • Related Report
      2012 Annual Research Report
  • [Presentation] 試験管内分子進化による高親和力抗エストラジオールscfvの創製と応用2012

    • Author(s)
      大山浩之,山口修子,中田茂利,丹羽俊文,小林典裕
    • Organizer
      第52回日本臨床化学会年次学術集会
    • Place of Presentation
      盛岡
    • Year and Date
      2012-09-07
    • Related Report
      2012 Annual Research Report 2012 Final Research Report
  • [Presentation] 実用診断試薬を創出する試験管内分子進化(3).抗エストラジオール変異scFvの諸性質2012

    • Author(s)
      大山浩之, 山口修子, 中田茂利, 小林典裕
    • Organizer
      日本薬学会第132年会
    • Place of Presentation
      札幌
    • Year and Date
      2012-03-29
    • Related Report
      2011 Annual Research Report
  • [Presentation] ss型抗イディオタイプ抗体scFv-酵素融合体を用いるハプテンイムノメトリックアッセイの試み2011

    • Author(s)
      大山浩之,田中瑛梨,丹羽俊文,小林典裕
    • Organizer
      日本分析化学会第60年会
    • Place of Presentation
      名古屋.
    • Year and Date
      2011-09-15
    • Related Report
      2012 Final Research Report
  • [Presentation] 部位特異的変異導入による抗コチニンscFvの試験管内親和性成熟の試み2011

    • Author(s)
      大山浩之,寺本隆佑,宮岡広子,森田いずみ,小林典裕
    • Organizer
      日本分析化学会第60年会
    • Place of Presentation
      名古屋
    • Year and Date
      2011-09-15
    • Related Report
      2012 Final Research Report 2011 Annual Research Report
  • [Presentation] β型抗イディオタイプ抗体scFv-酵素融合体を用いるハプテンイムノメトリックアッセイの試み2011

    • Author(s)
      大山浩之, 田中瑛梨, 丹羽俊文, 小林典裕
    • Organizer
      日本分析化学会第60年会
    • Place of Presentation
      名古屋
    • Year and Date
      2011-09-15
    • Related Report
      2011 Annual Research Report
  • [Presentation] 抗イディオタイプ抗体scFv-酵素融合体をトレーサーに用いる競合型ハプテンELISAの構築2011

    • Author(s)
      丹羽俊文,加賀幸斗,小嶋菜月,林慎一,大山浩之,田中瑛梨,小林典裕
    • Organizer
      日本薬学会第131年会
    • Place of Presentation
      静岡
    • Year and Date
      2011-03-29
    • Related Report
      2012 Final Research Report 2010 Annual Research Report
  • [Presentation] 高親和力抗ハプテン抗体の単離に用いるCleavableビオチン標識ハプテンの合成と性質2011

    • Author(s)
      森田いずみ,久保智士,大山浩之,小林典裕
    • Organizer
      日本薬学会第131年会
    • Place of Presentation
      静岡
    • Year and Date
      2011-03-29
    • Related Report
      2012 Final Research Report 2010 Annual Research Report
  • [Presentation] 実用診断試薬を創出する試験管内分子進化(1).抗エストラジオール抗体のKaが250倍も増大2011

    • Author(s)
      大山浩之,山口修子,綺田尚久,小林典裕
    • Organizer
      日本薬学会第131年会
    • Place of Presentation
      静岡
    • Year and Date
      2011-03-29
    • Related Report
      2012 Final Research Report 2010 Annual Research Report
  • [Presentation] 実用診断試薬を創出する試験管内分子進化(2).高感度受動喫煙モニタリングを目指す抗コチニン抗体の親和性成熟2011

    • Author(s)
      小林典裕,大山浩之,石井香好,久保智士,寺本隆祐,太田光煕
    • Organizer
      日本薬学会第131年会
    • Place of Presentation
      静岡
    • Year and Date
      2011-03-29
    • Related Report
      2012 Final Research Report 2010 Annual Research Report
  • [Presentation] 実用診断試薬を創出する試験管内分子進化(4).抗コチニン変異scFvの諸性質2011

    • Author(s)
      森田いずみ, 大山浩之, 石井香好, 渡部芳郎, 太田光煕, 小林典裕
    • Organizer
      日本薬学会第132年会
    • Place of Presentation
      札幌
    • Year and Date
      2011-03-29
    • Related Report
      2011 Annual Research Report
  • [Presentation] ELISAの高感度化を目的とする抗コチニン抗体の試験管内アフィニティマチュレーション2010

    • Author(s)
      大山浩之,番園恵理佳,石井香好,寺本隆佑,小林典裕,太田光煕
    • Organizer
      第50回日本臨床化学会年次学術集会
    • Place of Presentation
      甲府
    • Year and Date
      2010-09-24
    • Related Report
      2012 Final Research Report 2010 Annual Research Report
  • [Presentation] 抗体工学的アプローチによるエストラジオール免疫測定法の高感度化2010

    • Author(s)
      大山浩之,綺田尚久,山口修子,小林典裕
    • Organizer
      日本分析化学会第59年会
    • Place of Presentation
      仙台
    • Year and Date
      2010-09-15
    • Related Report
      2012 Final Research Report 2010 Annual Research Report
  • [Presentation] ハプテン・オリゴ糖複合体を認識するscFvの単離とハプテンイムノメトリックアッセイへの応用2010

    • Author(s)
      大山浩之,鈴木巌,安井孝治,小林典裕
    • Organizer
      日本分析化学会第59年会
    • Place of Presentation
      仙台
    • Year and Date
      2010-09-15
    • Related Report
      2012 Final Research Report 2010 Annual Research Report
  • [Presentation] Ka値が30倍以上増大:"第3世代"変異抗エストラジオール抗体フラグメント2010

    • Author(s)
      大山浩之,加藤芳徳,小林典裕
    • Organizer
      日本薬学会第130年会
    • Place of Presentation
      岡山
    • Year and Date
      2010-03-29
    • Related Report
      2012 Final Research Report
  • [Presentation] ヒト尿中コチニンELISA用モノクローナル抗体の調製とscFv化2010

    • Author(s)
      番園恵理佳,下駄祐子,国広俊臣,大山浩之,小林典裕,太田光煕
    • Organizer
      日本薬学会第130年会
    • Place of Presentation
      岡山
    • Year and Date
      2010-03-29
    • Related Report
      2012 Final Research Report
  • [Presentation] Generation of an engineered diagnostic antibody fragment for estradiol through 250-fold improvement in the affinity2010

    • Author(s)
      Hiroyuki Oyama
    • Organizer
      IBC 21st Annual International Conference: Antibody Engineering
    • Place of Presentation
      San Diego, USA.
    • Related Report
      2012 Final Research Report
  • [Presentation] Direct immunosensing of hapten molecules using single-chain Fv fragments that recognizehapten-oligosaccharide complexes2010

    • Author(s)
      Norihiro Kobayashi
    • Organizer
      IBC 21st Annual International Conference: Antibody Engineering
    • Place of Presentation
      San Diego, USA.
    • Related Report
      2012 Final Research Report
  • [Presentation] Generation of an engineered diagnostic antibody fragment for estradiol through 250-fold improvement in the affinity2010

    • Author(s)
      Hiroyuki Oyama
    • Organizer
      IBC 21st Annual International Conference : Antibody Engineering
    • Place of Presentation
      San Diego
    • Related Report
      2010 Annual Research Report
  • [Presentation] Direct immunosensing of hapten molecules using single-chain Fv fragments that recognize hapten-oligosaccharide complexes2010

    • Author(s)
      Norihiro Kobayashi
    • Organizer
      IBC 21st Annual International Conference : Antibody Engineering
    • Place of Presentation
      San Diego
    • Related Report
      2010 Annual Research Report
  • [Book] NEW薬品分析化学(第2版)2012

    • Author(s)
      小林典裕
    • Total Pages
      309
    • Publisher
      廣川書店
    • Related Report
      2012 Final Research Report
  • [Book] 薬学用語辞典2012

    • Author(s)
      日本薬学会編
    • Publisher
      東京化学同人
    • Related Report
      2012 Annual Research Report 2012 Final Research Report
  • [Book] スタンダード薬学シリーズ2・物理系薬学II,化学物質の分析(第3版)2012

    • Author(s)
      日本薬学会編
    • Publisher
      東京化学同人
    • Related Report
      2012 Final Research Report
  • [Book] NEW薬品分析化学 (第2版)2012

    • Author(s)
      小林 典裕
    • Total Pages
      309
    • Publisher
      廣川書店
    • Related Report
      2012 Annual Research Report
  • [Book] スタンダード薬学シリーズ2・物理系薬学II, 化学物質の分析 (第3版)2012

    • Author(s)
      日本薬学会編
    • Total Pages
      1
    • Publisher
      東京化学同人
    • Related Report
      2012 Annual Research Report
  • [Book] スタンダード薬学シリーズ2物理系薬学II.化学物質の分析2011

    • Author(s)
      日本薬学会編
    • Publisher
      東京化学同人
    • Related Report
      2012 Final Research Report
  • [Patent(Industrial Property Rights)] 核医学診断装置の制御方法,核医学診断装置,診断剤キット,および標識抗テネイシンC scFvフラグメント.2012

    • Inventor(s)
      小林典裕ほか11名
    • Industrial Property Number
      2012-102732
    • Filing Date
      2012
    • Related Report
      2012 Final Research Report

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Published: 2010-08-23   Modified: 2019-07-29  

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