Efficient generation of high-affinity mutated antibodies based on a single virion direct fluorescence detection of antibody-displaying phage
| Project/Area Number |
16K08954
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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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 |
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| Co-Investigator(Kenkyū-buntansha) |
森田 いずみ 神戸薬科大学, 薬学部, 助手 (20299085)
小林 典裕 神戸薬科大学, 薬学部, 教授 (90205477)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Project Status |
Completed (Fiscal Year 2018)
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| Budget Amount *help |
¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2018: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2017: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2016: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
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| Keywords | 抗体工学 / ファージ提示 / scFv / 共焦点レーザー顕微鏡 / 抗ファージ抗体 / タンパク質工学 / 免疫化学 / 進化分子工学 / 分析化学 |
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| Outline of Final Research Achievements |
Antibodies specifically bind to target antigen molecules, and thus are widely used as in vivo and in vitro diagnostic agents, and are essential functional molecules for clinical examination. Recently, generation of antibody mutants by antibody-engineering has attracted attention, but there are many problems with conventional selection methods (panning).
In this study, we have been succeeded in visualization of a single antigen-specific scFv-displaying phage using confocal laser fluorescence microscope. It will be possible to detect and isolate the improved clones in the scFv-displaying phage library "directly" under the confocal laser fluorescence microscope and it is expected that a drastic improvement of the phage display method will be made. Moreover, we developed into an array-type screening method and established a rapid and efficient isolation method for high affinity scFv-displaying phage.
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| Academic Significance and Societal Importance of the Research Achievements |
遺伝子操作による変異抗体の創製には抗体提示ファージライブラリー法が活用される。希少な改良型分子種を、大量のジャンク抗体から選別しつつ単離するため「試験管内分子進化」ともいわれるが、実用的な変異抗体の取得は容易ではない。疎水的なファージ粒子の性質や、ファージに提示される抗体分子の割合（提示率）が低いことが問題であった。本研究では、共焦点レーザー顕微鏡によってファージを可視化することで抗原特異的な抗体提示ファージを直接単離することが可能視された。また、これまで不明であった提示率を算出し、蛍光検出に用いた抗ファージ抗体によるアレイ型スクリーニング法を開発し、目的抗体の迅速な取得が期待される。
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Report
(4 results)
Research Products
(23 results)
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[Journal Article] Enantioselective Monoclonal Antibodies for Detecting Ketamine to Crack Down on Illicit Use2018
Author(s)
Morita, I. Oyama, H. Kanda, Y. Yasuo, M. Ito, A. Toyota, M. Hayashi, Y. Yokoyama, T. Kobayashi, N.
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Journal Title
Biological and Pharmaceutical Bulletin
Volume: 41
Issue: 1
Pages: 123-131
DOI
NAID
ISSN
0918-6158, 1347-5215
Related Report
Peer Reviewed / Open Access
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[Journal Article] One-Shot In Vitro Evolution Generated an Antibody Fragment for Testing Urinary Cotinine with More Than 40-Fold Enhanced Affinity2017
Author(s)
Oyama, Hiroyuki; Morita, Izumi; Kiguchi, Yuki; Banzono, Erika; Ishii, Kasumi; Kubo, Satoshi; Watanabe, Yoshiro; Hirai, Anna; Kaede, Chiaki; Ohta, Mitsuhiro; Kobayashi Norihiro
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Journal Title
Analytical Chemistry
Volume: 89(1)
Issue: 1
Pages: 988-995
DOI
Related Report
Peer Reviewed
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[Presentation] Effect of Heparin on Amyloid Fibril Formation of ApoA-I Fragment Peptides2016
Author(s)
Shiho Mikawa, Chiharu Mizuguchi, Izumi Morita, Hioryuki Oyama, Teruhiko Baba, Akira Shigenaga, Toshinori Shimanouchi, Norihiro Kobayashi, Akira Otaka, Kenichi Akaji, Hiroyuki Saito
Organizer
第53回ペプチド討論会
Place of Presentation
京都
Year and Date
2016-10-26
Related Report
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