| Project/Area Number |
17590504
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory medicine
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| Research Institution | Kobe Pharmaceutical University |
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Principal Investigator |
KOBAYASHI Norihiro Kobe Pharmaceutical University, Professor, 薬学部, 教授 (90205477)
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| Co-Investigator(Kenkyū-buntansha) |
KOYAMA Junko Kobe Pharmaceutical University, Lecturer, 薬学部, 講師 (60102109)
MORITA Mimi Kobe Pharmaceutical University, Assistant Professor, 薬学部, 助手 (20299085)
KATO Yoshinori Kobe Pharmaceutical University, Assistant Professor, 薬学部, 助手 (10368491)
GOTO Junichi Tohoku University Hospital, Professor, 病院・教授 (80006337)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | hapten / acceptor / complex / antibody / immunosensing / cyclodextrin / single-domain antibody / antibody-engineering / アクセプター / 一本鎖Fvフラグメント / 抗体ライブラリー |
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| Research Abstract |
Trace characterization of physiologically active small molecules (haptens) is an important subject in biomedical analysis. Unfortunately, noncompetitive two-site immunometric assays are not applicable to the haptens, because of their low molecular mass, resulting in the difficulty in gaining subfemtomole sensitivity. To overcome this limitation, we planed a new approach employing the small molecules showing an affinity to the target haptens (acceptors; ACC) and engineered antibodies specific to the hapten-ACC complexes : such antibodies are expected to enable highly sensitive immunometric-type sensing systems for haptens.
β-Cyclodextrin (β-CD) was selected as the ACC, and 25-hydroxyvitaminD_3, 9-cis-retinoic acid and phenolphthalein (PP) were as model haptens. We isolated the phage clones displaying the single-chain Fv fragments (scFvs) that exhibit ca. 2〜4-fold stronger binding in the presence of hapten than the absence for each hapten from a mutated scFv-displaying phage library. One of these clones selected against PP afforded increasing signal intensity with the increase in the amount of added PP (ranging from 4 to 100 nmol) suggesting the availability of the displayed scFv for the expected hapten immunosensing systems. We then attempted to use the single-domain antibody (sdAb ; the antibody fragments that consist of only V_H domain) as ACC. A library of mutated V_H domains derived from a previously established mouse anti-estradiol (E_2) antibody was constructed, and some clones showing the binding to E_2 immobilized on microplate were isolated. We are now planning to create the improved sdAbs showing higher affinity to E_2 by introducing further point mutations. Because sdAbs would possess higher a affinity and specificity to a target than CDs, sdAbs are expected to be more practical ACC in the immunosensing systems.
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