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

开发“异质捕获夹心EIA”并检测激素干扰物质

基本信息

  • 批准号:
    17590281
  • 负责人:
  • 金额:
    $ 2.3万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
  • 财政年份:
    2005
  • 资助国家:
    日本
  • 起止时间:
    2005 至 2006
  • 项目状态:
    已结题

项目摘要

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.
我们开发了一种新的超灵敏的免疫测定“异捕获夹心EIA”,在amol水平检测等于或小于分子量1KD的半抗原,包括激素干扰物质,能够高灵敏度检测低分子物质。因此,采用噬菌体展示法,对一抗与低分子抗原的复合物制备第二抗体,并利用这两种抗体建立超灵敏非竞争方法。通过检测对蛋白质降解机制的影响,分析激素干扰物质对人体的影响。作为模型半抗原,使用E-64c。该E-64c在体内用巯基蛋白酶抑制剂强烈抑制溶酶体组织蛋白酶B。因此,利用E-64c很好地阐明了胞内降解机制,但其代谢机制尚不明确。在“异捕获夹心EIA”中,我们将E-64c与一抗反应,形成E-64c抗体复合物。使用e -64c抗体结合位点的特异性第二抗体连续测量该复合物。因此采用“噬菌体展示法”制备特异性二抗,但一抗必须为单克隆抗体。然后我们首先决定制备E-64c的单克隆抗体。用碳二亚胺制备E-64c-KLH,免疫小鼠,制备单克隆抗体。目前,我们正在利用噬菌体展示法制备该单克隆抗体/ E-64c结合位点特异性的人工抗体。当我们同时使用两种标记抗体时,我们研究了各种标记化合物和酶标记来逃避位阻。结果表明,从代谢周转率和分子量来看,重组碱性磷酸酶是最合适的,偶联研究认为,马来酰亚胺-铰链法是最合适的。

项目成果

期刊论文数量(14)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest
  • DOI:
    10.1002/mus.20611
  • 发表时间:
    2006-10-01
  • 期刊:
  • 影响因子:
    3.4
  • 作者:
    Ogawa, Takayuki;Furochi, Harumi;Nikawa, Takeshi
  • 通讯作者:
    Nikawa, Takeshi
Adrenomedullin release in the rat mesenteric resistance artery
大鼠肠系膜阻力动脉中肾上腺髓质素的释放
  • DOI:
  • 发表时间:
    2005
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Akiyama S;Hobara N;Maruo N;Hashida S;Kitamura K;Eto T.;Kawasaki H.
  • 通讯作者:
    Kawasaki H.
Ubiquitin ligase gene expression in healthy volenteers with 20-days bed rest.
卧床休息 20 天的健康志愿者中泛素连接酶基因的表达。
  • DOI:
  • 发表时间:
    2006
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Iwabuchi;K. et al.;Ogawa T
  • 通讯作者:
    Ogawa T
Adrenomedullin release in the rat mesenteric resistance artery.
大鼠肠系膜阻力动脉中肾上腺髓质素的释放。
  • DOI:
  • 发表时间:
    2005
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Nojima K;Hochegger H;Saberi A;Fukushima T;Kikuchi K;Yoshimura M;Orelli BJ;Bishop DK;Hirano S;Ohzeki M;Ishiai M;Yamamoto K;Takata M;Arakawa H;Buerstedde JM;Yamazoe M;Kawamoto T;Araki K;Takahashi JA;Hashimoto N;Takeda S;Sonoda E.;Akiyama S.
  • 通讯作者:
    Akiyama S.
Plasma intact ghrelin levels in obese children.
肥胖儿童血浆完整生长素释放肽水平。
  • DOI:
  • 发表时间:
    2006
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Kitao H;Yamamoto K;etal.;Ikeda R;Jo Satoh;Takae Minami;Inoue S.
  • 通讯作者:
    Inoue S.
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HASHIDA Seiichi其他文献

HASHIDA Seiichi的其他文献

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{{ truncateString('HASHIDA Seiichi', 18)}}的其他基金

Development of a diabetes risk profile using a urinary biomarker and its application in the assessment of nutrient status and exercise levels.
使用尿液生物标志物开发糖尿病风险概况及其在营养状况和运动水平评估中的应用。
  • 批准号:
    21500701
  • 财政年份:
    2009
  • 资助金额:
    $ 2.3万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Development of novel ultra sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for provirus DNA and virus RNA of HIV-1 (Early diagnosis of HIV-1 infection)
开发新型超灵敏酶联免疫分析法(免疫复合物转移酶联免疫分析法)检测HIV-1原病毒DNA和病毒RNA(HIV-1感染的早期诊断)
  • 批准号:
    12672247
  • 财政年份:
    2000
  • 资助金额:
    $ 2.3万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Studies for the Practical Use of Novel and Ultrasensitive Enzyme Immunoassay (Immune Complex Transfer Enzyme Immunoassay) of Anti-HTLV-I IgG Using Synthetic Peptides as Antigens
以合成肽为抗原的抗 HTLV-I IgG 新型超灵敏酶免疫分析(免疫复合物转移酶免疫分析)的实际应用研究
  • 批准号:
    08670154
  • 财政年份:
    1996
  • 资助金额:
    $ 2.3万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Basic Study on the Development and Application of Novel and Ultrasensitive Immunoassay for Haptens, Especially, Small Peptide Molecules
半抗原特别是小肽分子新型超灵敏免疫分析技术的开发和应用基础研究
  • 批准号:
    02808029
  • 财政年份:
    1990
  • 资助金额:
    $ 2.3万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
Basic Study on Development and Application of Ultrasensitive Enzyme Immunoassay for Interleukins
白细胞介素超灵敏酶联免疫分析法的开发及应用基础研究
  • 批准号:
    63580125
  • 财政年份:
    1988
  • 资助金额:
    $ 2.3万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)

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