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Development of "heterocapture sandwich EIA" and detection of the a hormone-disrupting substance

Research Project

Project/Area Number 17590281
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Pathological medical chemistry
Research InstitutionTokushima Bunri University

Principal Investigator

HASHIDA Seiichi  Tokushima Bunri University, Institute for Health Sciences, Professor, 健康科学研究所, 教授 (10156268)

Co-Investigator(Kenkyū-buntansha) ISHIDOH Kazumi  Tokushima Bunri University, Institute for Health Sciences, Professor, 健康科学研究所, 教授 (40212906)
Project Period (FY) 2005 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2005: ¥2,900,000 (Direct Cost: ¥2,900,000)
KeywordsHapten / Immunoassay / phage display method / Monoclonal antibody / ELISA / 抗体 / 酵素 / 分析化学 / 内分泌撹乱物質
Research Abstract

We develop new ultra sensitive immunoassay "heterocapture sandwich EIA" detecting a haptenic antigen equal to or less than molecular weight 1KD at an amol level and, including a hormone-disrupting substance, enable high sensitive detection of the low-molecular substance. Therefore, using a phage display method, it prepares second antibody for the complex of the primary antibody with a low molecule antigen and establishes ultra sensitive non-competitive method by these two kinds of antibodies. We examined the influence on protein degradation mechanism to analyze the influence on human body by the a hormone-disrupting substance.
As a model hapten, it was used E-64c. This E-64c strongly inhibits cathepsin B in lysosome with thiol protease inhibitor in vivo. Therefore, E-64c is used for the elucidation of the intracellular degradation mechanism well, but the metabolism mechanism is not apparent. In "the heterocapture sandwich EIA", we react E-64c and primary antibody and form an E-64c-antibody complex. This complex is measured using specific second antibody for the E-64c-antibody combining site continuously. So it prepares specific second antibody by "a phage display method", but primary antibody must be monoclonal antibody. Then at first we decided to prepare monoclonal antibody for E-64c. We made E-64c-KLH using carbodiimide and immunized it to mouse and prepared monoclonal antibody. Now, we are making the artificial antibody which is specific for this monoclonal antibody / E-64c binding site by a phage display method.
We examined various kinds of labeled compounds and enzymatic tagging to evade steric hindrance when we used two kinds of labeled antibody in parallel with this. As a result, recombinant alkaline phosphatase was suitable from a metabolic turnover rate and molecular weight, and conjugation understood that a maleimide-hinge method was suitable most.

Report

(3 results)
  • 2006 Annual Research Report   Final Research Report Summary
  • 2005 Annual Research Report
  • Research Products

    (14 results)

All 2006 2005 Other

All Journal Article (14 results)

  • [Journal Article] Plasma intact ghrelin levels in obese children.2006

    • Author(s)
      Inoue S.
    • Journal Title

      Growth Hormone & IGF Research 16

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Ubiquitinligase Cbl-b downregulates bone formation through suppression of IGF-I signaling in osteoblasts during denervation.2006

    • Author(s)
      Suzue N
    • Journal Title

      J. Bone Mineral Res. 21

      Pages: 722-734

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Ubiquitin ligase gene expression in healthy volenteers with 20-day bed rest.2006

    • Author(s)
      Ogawa T
    • Journal Title

      Muscle Nerve 34

      Pages: 463-469

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Plasma intact ghrelin levels in obese children.2006

    • Author(s)
      Inoue S., Hashida S.
    • Journal Title

      Growth Hormone & IGF Research. 16

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Ubiquitinligase Cbl-b downregulates bone formation through suppression of IGF-I signaling in osteoblasts during denervation.2006

    • Author(s)
      Suzue N, Nikawa T, Onishi Y, Yamada C, Hirasaka K, Ogawa T, Furochi H, Kosaka H, Ishidoh K, Gu H, Takeda S, Ishimaru N, Hayashi Y, Yamamoto H, Kishi K, Yasui N.
    • Journal Title

      J.Bone Mineral Res. 21

      Pages: 722-734

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Ubiquitin ligase gene expression in healthy volunteers with 20-day bed rest.2006

    • Author(s)
      Ogawa T, Furochi H, Mameoka M, Hirasaka K, Onishi Y, Suzue N, Oarada M, Akamatsu M, Akima H, Fukunaga T, Kishi K, Yasui N, Ishidoh K, Fukuoka H, Nikawa T.
    • Journal Title

      Muscle Nerve 34

      Pages: 463-469

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Plasma intact ghrelin levels in obese children2006

    • Author(s)
      Inoue S.
    • Journal Title

      Growth Hormone & IGF Research 16

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Ubiquitin ligase gene expression in healthy volenteers with 20-days bed rest.2006

    • Author(s)
      Ogawa T
    • Journal Title

      Muscle Nerve 34

      Pages: 463-469

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Adrenomedullin release in the rat mesenteric resistance artery.2005

    • Author(s)
      Akiyama S.
    • Journal Title

      Peptides 26

      Pages: 2222-2230

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Study of blood metabolism and urinary excretion of chymopapain following intradiscal injection using a high-sensitive enzyme immunoassay.2005

    • Author(s)
      Kawano K.
    • Journal Title

      J Orthop Sci. 10

      Pages: 214-220

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Study of blood metabolism and urinary excretion of chymopapain following intradiscal injection using a high-sensitivity enzyme immunoassay.2005

    • Author(s)
      Kawano K, Tajima N, Hashida S, Ishikawa E.
    • Journal Title

      J.Orthopaedic Sci. 10

      Pages: 206-213

    • NAID

      10015564283

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Adrenomedullin release in the rat mesenteric resistance artery2005

    • Author(s)
      Akiyama S, Hobara N, Maruo N, Hashida S, Kitamura K, Eto T., Kawasaki H.
    • Journal Title

      Peptides. 26

      Pages: 2222-2230

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Adrenomedullin release in the rat mesenteric resistance artery2005

    • Author(s)
      Akiyama S.
    • Journal Title

      Peptides 26

      Pages: 2222-2230

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Ubiquitinligase Cbl-6 downregulates bone formation through suppression of IGF-I signaling in osteoblasts during denervation.

    • Author(s)
      Suzue N
    • Journal Title

      J.bone Mineral Res.

    • Related Report
      2006 Annual Research Report

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Published: 2005-04-01   Modified: 2016-04-21  

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