| Project/Area Number |
17360394
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biofunction/Bioprocess
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| Research Institution | The University of Tokyo |
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Principal Investigator |
UEDA Hiroshi The University of Tokyo, School of Engineering, Associate Professor (60232758)
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| Co-Investigator(Kenkyū-buntansha) |
IHARA Masaki University of Tokyo, School of Engineering, Assistant Professor (50391868)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥12,770,000 (Direct Cost: ¥11,900,000、Indirect Cost: ¥870,000)
Fiscal Year 2007: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000)
Fiscal Year 2006: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2005: ¥6,200,000 (Direct Cost: ¥6,200,000)
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| Keywords | phage display / immunoassay / antibody variable region / open sandwich / mycotoxin / ELISA / peptide / trans splicing / アルカリフォスファターゼ / サンドイッチ法 / 免役測定 / スプライシング / Phage display / Immunoassay / Variable region / alkaline phosphatase / luciferase / open sandwich |
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| Research Abstract |
The objective of this study was to make a comprehensive and sensitive detection system of low molecular weight analytes through the use of a novel immunoassay principle that utilizes the stabilization of antibody Fv region upon binding with antigen (open sandwich immunoassay, OS-IA). To this end, we cloned the Fv genes of the antibodies for two anti-neonicotinoid pesticides, a mycotoxin, and a low molecular weight peptide BGP-C7, and succeeded in the construction of OS-IA system and sensitive noncompetitive detection thereof. Especially in the last system, rapid detection from a minute amount of serum sample was attained by a microchip-based detection system.
Through this process, we realized the importance of optimized phage display format for the selection of antibodies that are suitable to OS-IA. In addition to split Fv system already developed, an scFv-OS conversion system based on ere recombinase was developed, as well as an Fab-OS conversion system which enabled construction and selection of larger library of 10^8 size. While the scheduled construction of cell-free selection system is yet to be done, we think efficient selection of larger and focused library for OS-IA is soon to be realized, which will enable sensitive detection of various small analytes such as environmental pollutants and protein modification such as phosphorylation and truncation.
As an unplanned result, we developed a novel method to express antibody fusion proteins in antibody producing cells through ''trans'' splicing of the pre-mRNAs in the cell nucleus. As a model system, a VH-enzyme fusion mRNA and corresponding protein that is indispensable in OS-IA were successfully expressed in COS-1 cells. Elucidation of rapid fabrication methods of such fusion proteins from established hybridoma resources without the need of gene cloning is envisaged based on this principle as well as other principles.
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