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Generation of "antibody-like" proteins based on evolutionary molecular engineering for novel clinical diagnostics

Research Project

Project/Area Number 25460709
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Research Field Laboratory medicine
Research InstitutionKobe Pharmaceutical University

Principal Investigator

MORITA IZUMI  神戸薬科大学, 薬学部, 助手 (20299085)

Co-Investigator(Kenkyū-buntansha) KOBAYASHI Norihiro  神戸薬科大学, 薬学部, 教授 (90205477)
OYAMA Hiroyuki  神戸薬科大学, 薬学部, 助教 (80572966)
Project Period (FY) 2013-04-01 – 2016-03-31
Project Status Completed (Fiscal Year 2015)
Budget Amount *help
¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000)
Fiscal Year 2015: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2013: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Keywords進化分子工学
Outline of Final Research Achievements

Antibodies are widely used in various clinical tests. However, lower expression levels in E. coli and their nature requiring two variable domains (VH and VL) for building the antigen-binding sites reduce their utility in genetic manipulation for generating improved species. Therefore, we attempted creation of new "antibody-like" proteins, which are composed of a single domain scaffold with much smaller molecular weight and can be expressed with a satisfactory yield in E. coli. We chose the biotin-binding protein, streptavidin monomer (stav'), as the basic scaffold. After several steps of mutagenesis and selection for their loop structures protruding from the scaffold, we succeeded in obtaining stav' mutants that had gained novel binding specificities targeting a steroid, estradiol.

Report

(4 results)
  • 2015 Annual Research Report   Final Research Report ( PDF )
  • 2014 Research-status Report
  • 2013 Research-status Report
  • Research Products

    (33 results)

All 2016 2015 2014 2013 Other

All Journal Article (6 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 6 results,  Acknowledgement Compliant: 1 results) Presentation (23 results) (of which Int'l Joint Research: 3 results,  Invited: 1 results) Book (2 results) Remarks (2 results)

  • [Journal Article] Gaussia Luciferase as a Genetic Fusion Partner with Antibody Fragments for Sensitive Immunoassay Monitoring of Clinical Biomarkers2015

    • Author(s)
      Oyama Hiroyuki; Morita Izumi; Kiguchi Yuki; Miyake Sayaka; Moriuchi Ayaka; Akisada Tatsuki; Kobayashi Norihiro; Niwa Toshifumi
    • Journal Title

      Analytical chemistry

      Volume: 87(24) Issue: 24 Pages: 12387-12395

    • DOI

      10.1021/acs.analchem.5b04015

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Two chalcones, 4-hydroxyderricin and xanthoangelol, stimulate GLUT4-dependent glucose uptake through the LKB1/AMP-activated protein kinase signaling pathway in 3T3-L1 adipocytes2015

    • Author(s)
      Ohta, Mitsuhiro; Fujinami, Aya; Kobayashi, Norihiro; Amano, Akiko; Ishigami, Akihito; Tokuda, Harukuni; Suzuki, Nobutaka; Ito, Fumitake; Mori, Taisuke; Sawada, Morio;
    • Journal Title

      Nutrition Research

      Volume: 35(7) Issue: 7 Pages: 618-625

    • DOI

      10.1016/j.nutres.2015.05.010

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Cellular Interaction and Cytotoxicity of the Iowa Mutation of Apolipoprotein A-I (ApoA-IIowa) Amyloid Mediated by Sulfate Moieties of Heparan Sulfate2015

    • Author(s)
      Kuwabara, K., Nishitsuji, K., Uchimura, K., Hung, S.C., Mizuguchi, M., Nakajima, H., Mikawa, S., Kobayashi, N., Saito, H., and Sakashita, N.
    • Journal Title

      J Biol Chem

      Volume: 290 Issue: 40 Pages: 24210-24221

    • DOI

      10.1074/jbc.m115.652545

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Direct detection of ABCA1-dependent HDL formation based on lipidation-induced hydrophobicity change in apoA-I.2014

    • Author(s)
      Risa Omura, Kohjiro Nagao, Norihiro Kobayashi, Kazumitsu Ueda, and Hiroyuki Saito
    • Journal Title

      J Lipid Res.

      Volume: 55 Issue: 11 Pages: 2423-2431

    • DOI

      10.1194/jlr.d049445

    • Related Report
      2014 Research-status Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Anti-idiotype scFv-enzyme fusion proteins: A clonable analyte-mimicking probe for standardized immunoassays targeting small biomarkers.2013

    • Author(s)
      Oyama H., Tanaka E., Kawanaka T., Morita I., Niwa T., Kobayashi N.
    • Journal Title

      Analytical Chemistry

      Volume: 85 Issue: 23 Pages: 11553-11559

    • DOI

      10.1021/ac402868f

    • Related Report
      2013 Research-status Report
    • Peer Reviewed
  • [Journal Article] “Breeding” diagnostic antibodies for higher assay performance: a 250-fold affinity-matured antibody mutant targeting a small biomarker.2013

    • Author(s)
      Oyama H., Yamaguchi S., Nakata S., Niwa T., Kobayashi N.
    • Journal Title

      Analytical Chemistry

      Volume: 85 Issue: 10 Pages: 4930-4937

    • DOI

      10.1021/ac3037602

    • Related Report
      2013 Research-status Report
    • Peer Reviewed
  • [Presentation] オンサイト分析を目的とする Teoc 化覚せいアミンに対するモノクローナル抗体の作製2016

    • Author(s)
      黒田 裕美, 森田 いずみ, 伊藤 綾, 小山 千尋, 池田 夏美, 大山 浩之, 小林 典裕
    • Organizer
      日本薬学会第136年会
    • Place of Presentation
      横浜
    • Year and Date
      2016-03-28
    • Related Report
      2015 Annual Research Report
  • [Presentation] 抗エストラジオールscFv 試験管内親和性成熟機構の解析(2)2016

    • Author(s)
      山本 知佳, 大山 浩之, 木口 裕貴, 森田 いずみ, 小林 典裕
    • Organizer
      日本薬学会第136年会
    • Place of Presentation
      横浜
    • Year and Date
      2016-03-28
    • Related Report
      2015 Annual Research Report
  • [Presentation] 高性能変異抗体の効率的単離を目的とする抗ファージ scFv-GLuc 融合体の調製2016

    • Author(s)
      木口 裕貴, 藤田 真聡, 片山 恵美子, 大山 浩之, 森田 いずみ, 小林 典裕
    • Organizer
      日本薬学会第136年会
    • Place of Presentation
      横浜
    • Year and Date
      2016-03-28
    • Related Report
      2015 Annual Research Report
  • [Presentation] 抗コルチゾールscFv の親和性成熟における部位特異的点変異の効果2016

    • Author(s)
      大山 浩之, 徳重 仁美, 知念 絵美理, 森下 知美, 森田 いずみ, 小林 典裕
    • Organizer
      日本薬学会第136年会
    • Place of Presentation
      横浜
    • Year and Date
      2016-03-28
    • Related Report
      2015 Annual Research Report
  • [Presentation] 試験管内親和性成熟による実用抗体の創製:低分子バイオマーカーを例に2015

    • Author(s)
      大山 浩之
    • Organizer
      生物化学的測定研究会第20回学術シンポジウム
    • Place of Presentation
      神戸
    • Year and Date
      2015-11-13
    • Related Report
      2015 Annual Research Report
    • Invited
  • [Presentation] 高親和力抗エストラジオール変異scFvの創製とその親和性成熟機構の解析2015

    • Author(s)
      大山 浩之,江浪 友理,田川 達矢,山本 知佳,森田 いずみ,小林 典裕
    • Organizer
      第55回日本臨床化学会年次学術集会
    • Place of Presentation
      吹田
    • Year and Date
      2015-10-31
    • Related Report
      2015 Annual Research Report
  • [Presentation] 改良型抗エストラジオールscFvの親和性成熟機構の解析2015

    • Author(s)
      大山 浩之,江浪 友理, 田川 達矢,山本 知佳,森田 いずみ,小林 典裕
    • Organizer
      日本分析化学会第64年会
    • Place of Presentation
      福岡
    • Year and Date
      2015-09-11
    • Related Report
      2015 Annual Research Report
  • [Presentation] scFv-ルシフェラーゼ融合タンパク質を用いるチロキシン生物発光ELISAの試み2015

    • Author(s)
      大山 浩之,宮下 貴之,森田 いずみ,小林 典裕
    • Organizer
      日本分析化学会第64年会
    • Place of Presentation
      福岡
    • Year and Date
      2015-09-11
    • Related Report
      2015 Annual Research Report
  • [Presentation] Sensitive luminescent ELISA for human serum cortisol using a fusion protein combining anti-cortisol scFv and Gaussia lusiferase2015

    • Author(s)
      Hiroyuki Oyama, Norihiro Kobayashi
    • Organizer
      European Congress of Clinical Chemistry and Laboratory Medicine
    • Place of Presentation
      paris
    • Year and Date
      2015-06-22
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] "Antibody breeding" for more sensitive immunoassays 1: Three-step affinity maturarion generated an improved scFv suitable for serum estradiol-17 beta ELISA2015

    • Author(s)
      Hiroyuki Oyama, Norihiro Kobayashi
    • Organizer
      European Congress of Clinical Chemistry and Laboratory Medicine
    • Place of Presentation
      paris
    • Year and Date
      2015-06-22
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] "Antibody breeding" for more sensitive immunoassays 2: Human urinary cotinine ELISA using an affinity matured scFv to monitor tabacco smoke exposure2015

    • Author(s)
      Izumi Morita, Hiroyuki Oyama, Norihiro Kobayashi
    • Organizer
      European Congress of Clinical Chemistry and Laboratory Medicine
    • Place of Presentation
      paris
    • Year and Date
      2015-06-22
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] 抗コチニン scFv の親和性成熟における部位特異的点変異の効果2015

    • Author(s)
      森田 いずみ, 西村 沙貴, 蓑田 早耶香, 木口 裕貴, 大山 浩之, 小林 典裕
    • Organizer
      日本薬学会第135年会
    • Place of Presentation
      神戸
    • Year and Date
      2015-03-27
    • Related Report
      2014 Research-status Report
  • [Presentation] 大麻成分に対するモノクローナル抗体の調製とキャラクタリゼーション2015

    • Author(s)
      伊藤 綾, 安尾 まゆみ, 竜田 都加, 森田 いずみ, 大山 浩之, 小林 典裕
    • Organizer
      日本薬学会第135年会
    • Place of Presentation
      神戸
    • Year and Date
      2015-03-27
    • Related Report
      2014 Research-status Report
  • [Presentation] 抗エストラジオール scFv 試験管内親和性成熟機構の解析2015

    • Author(s)
      大山 浩之, 江浪 友理, 木口 裕貴, 森田 いずみ, 小林 典裕
    • Organizer
      日本薬学会第135年会
    • Place of Presentation
      神戸
    • Year and Date
      2015-03-27
    • Related Report
      2014 Research-status Report
  • [Presentation] 改良型変異scFvフラグメントを用いるヒト尿中コチニンのモノクローナルELISA2014

    • Author(s)
      森田いずみ、平井杏奈、西村沙貴、大山浩之、太田光煕、小林典裕
    • Organizer
      日本分析化学会第63年会
    • Place of Presentation
      東広島
    • Year and Date
      2014-09-18
    • Related Report
      2014 Research-status Report
  • [Presentation] 抗チロキシン一本鎖 Fvフラグメントの創製と諸性質2014

    • Author(s)
      森田 いずみ,寺澤 清伽,泉 亜紀,宮岡 広子,大山 浩之,小林 典裕
    • Organizer
      日本薬学会第134年会
    • Place of Presentation
      熊本
    • Related Report
      2013 Research-status Report
  • [Presentation] 実用診断試薬を創出する試験管内分子進化 (7). 低親和力抗コルチゾール抗体の親 和性成熟2014

    • Author(s)
      大山 浩之,森下 知美,福島 咲子,安井 孝治,小林 典裕
    • Organizer
      日本薬学会第134年会
    • Place of Presentation
      熊本
    • Related Report
      2013 Research-status Report
  • [Presentation] scFv― ルシフェラーゼ融合タンパク質を用いるコルチゾール高感度発光 ELISA の開発2014

    • Author(s)
      大山 浩之,三宅 沙也香,秋定 辰紀,森内 彩香,丹羽 俊文, 小林 典裕
    • Organizer
      日本薬学会第134年会
    • Place of Presentation
      熊本
    • Related Report
      2013 Research-status Report
  • [Presentation] ""Breeding" Diagnostic Antibodies: An Affinity-Matured scFv for Urinary Cotinine ELISA to Monitor Tobacco Smoke Exposure2013

    • Author(s)
      Morita I., Oyama H., Banzono E., Ohta M., Kobayashi N.
    • Organizer
      Fifth Annual PEGS Europe : Protein & Antibody Engineering Summit
    • Place of Presentation
      Lisbon
    • Related Report
      2013 Research-status Report
  • [Presentation] 試験管内親和性成熟scFvを用いるヒト尿中コチニンのELISA2013

    • Author(s)
      大山 浩之,森田 いずみ,太田 光熙,小林 典裕
    • Organizer
      生物化学的測定研究会 第18回学術集会・総会
    • Place of Presentation
      京都
    • Related Report
      2013 Research-status Report
  • [Presentation] 高親和力scFv融合タンパク質を用いるコルチゾールの高感度ELISA2013

    • Author(s)
      大山 浩之,森田 いずみ,太田 光熙,小林 典裕
    • Organizer
      第53回日本臨床化学会年次学術集会
    • Place of Presentation
      徳島
    • Related Report
      2013 Research-status Report
  • [Presentation] Anti-Idiotype scFv-Enzyme Fusion Proteins Work as a Clonable Analyte-Mimicking Probe That Enables Standardization of Hapten Immunoassays2013

    • Author(s)
      Kobayashi N., Oyama H., Morita I., Niwa T.
    • Organizer
      Fifth Annual PEGS Europe : Protein & Antibody Engineering Summit
    • Place of Presentation
      Lisbon
    • Related Report
      2013 Research-status Report
  • [Presentation] "Breeding" Diagnostic Antibodies: A 250-Fold Affinity-Matured scFv Mutant for Measuring Human Serum Estradiol-17β2013

    • Author(s)
      Oyama H., Niwa T., Kobayashi N.
    • Organizer
      Fifth Annual PEGS Europe : Protein & Antibody Engineering Summit
    • Place of Presentation
      Lisbon
    • Related Report
      2013 Research-status Report
  • [Book] 免疫測定法 基礎から先端まで2015

    • Author(s)
      生物化学測定研究会 編
    • Total Pages
      325
    • Publisher
      講談社
    • Related Report
      2015 Annual Research Report
  • [Book] 免疫測定法2014

    • Author(s)
      生物化学的測定研究会編
    • Total Pages
      323
    • Publisher
      講談社
    • Related Report
      2014 Research-status Report
  • [Remarks] 神戸薬科大学 生命分析化学研究室

    • URL

      http://www.kobepharma-u.ac.jp/analchem/

    • Related Report
      2015 Annual Research Report
  • [Remarks] 神戸薬科大学 生命分析化学研究室ホームページ

    • URL

      http://www.kobepharma-u.ac.jp/analchem/

    • Related Report
      2014 Research-status Report

URL: 

Published: 2014-07-25   Modified: 2019-07-29  

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