Depelopment of immunochemical fluorescent sensor on the basis of tripartite split-GFP

• Project/Area Number: 17K15466
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Natural medicines
• Research Institution: Kyushu University
• Principal Investigator: Sakamoto Seiichi 九州大学, 薬学研究院, 准教授 (50610177)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2019: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: 小型化抗体 / ハリントニン / ELISA / 蛍光センサー / 三分割GFP

Outline of Final Research Achievements

This study aimed to develop rapid and sensitive immunological fluorescent sensor that can determine harringtonine (HT).
Primarily, VH-GFP10, GFP11-VL, and GFP1-9 genes were constructed to detect fluorescence in the presence of HT. However, the fluorescence derived from quaternary complex was not detected using this system. Subsequently, VH-GFP10-GFP11-VL (scFvGs) gene was constructed, in which VH-GFP10 and GFP11-VL was joined via flexible linker peptides of (GGGGS)n(n=2, 3). When the activity of scFvGs was investigated by using ELISA, it showed specific competitive activity to HT. Therefore, scFvGs were applied to immunological fluorescent sensor to detect fluorescence. As a result, slight fluorescence was detected compared with control. However, it was too weak to apply to quantitative analysis, suggesting that more optimization is required for development of this system.

Academic Significance and Societal Importance of the Research Achievements

本研究における対象化合物は低分子化合物であることから、二つの波及効果が考えられる。
一つは、違法薬物問題への貢献である。近年、社会問題となっている違法薬物は、その大部分が低分子化合物である。そのため、対象化合物HTを危険ドラッグや麻薬関連化合物へ置き換える事で社会問題となっている違法薬物の簡易迅速検出法へと応用できる。もう一つは、医薬品シーズ探索研究への貢献である。本システムの抗体遺伝子を他の受容体や疾病関連タンパク質に置き換えることで、無限の可能性を有する薬用資源から網羅的なスクリーニングが行なえ医薬品シーズの探索が可能となる。
本研究は、違法薬物問題や希少疾病医薬品開発問題の解決にも繋がる。

Research Products

• [Int'l Joint Research] Khon Kaen University/Walailak University/Mahidol University(タイ)
• [Journal Article] An estimated quantitative lateral flow immunoassay for determination of artesunate using monoclonal antibody (2020)
  • Author(s): Kitisripanya, T., Sermpradit, W., Sakamoto, S., Tanaka, H., Putalun, W.
  • Journal Title: Biomedical Chromatography Volume: 34 Issue: 2
  • DOI: 10.1002/bmc.4718
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Development of a simple and rapid method for detection of isomiroestrol in Pueraria candollei by an immunochromatographic strip test (2019)
  • Author(s): Chaingam, J., Kitisripanya, T., Krittanai, S., Sakamoto, S., Tanaka, H., Putalun, W.
  • Journal Title: Journanl of Natural Medicines Volume: 73 Issue: 3 Pages: 577-583
  • DOI: 10.1007/s11418-019-01307-6
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Modification of first constant domain of heavy chain (CH1) enabled effective folding of functional anti-forskolin antigen-binding fragment (Fab) for sensitive quantitative analysis (2019)
  • Author(s): Yusakul, G., Sakamoto, S., Tanaka, H., Morimoto, S.
  • Journal Title: Biotechnology Progress Volume: 35 Issue: 4
  • DOI: 10.1002/btpr.2822
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Preparation of a highly specific single chain variable fragment antibody targeting miroestrol and its application in quality control of Pueraria candollei by enzyme-linked immunosorbent assay (2019)
  • Author(s): Pongkitwitoon, B., Boonsnongcheep, P., Kitisripanya, T., Yusakul, G., Sakamoto, S., Tanaka, H., Morimoto, S., Putalun, W.
  • Journal Title: Phytochemical Analysis Volume: 30 Issue: 6 Pages: 600-608
  • DOI: 10.1002/pca.2832
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Preincubation format for a sensitive immunochromatographic assay for monocrotaline, a toxic pyrrolizidine alkaloid (2019)
  • Author(s): Yusakul, G., Sakamoto, S., Chanpokapaiboon, K., Tanaka, H., Morimoto, S.
  • Journal Title: Phytochemical Analysis Volume: 30 Issue: 6 Pages: 653-660
  • DOI: 10.1002/pca.2838
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Sodium-periodate mediated harringtonine derivatives and their antiproliferative activity against HL-60 acute leukemia cells (2018)
  • Author(s): Sakamoto, S., Miyamoto, T., Usui, K., Tanaka, H., Morimoto, S.
  • Journal Title: Journal of Natural Products Volume: 81 Issue: 1 Pages: 34-40
  • DOI: 10.1021/acs.jnatprod.7b00541
  • Peer Reviewed
• [Journal Article] Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites (2018)
  • Author(s): Sakamoto, S., Putalun, W., Vimolmangkang, S., Phoolcharoen, W., Shoyama, Y., Tanaka, H., Morimoto, S.
  • Journal Title: Journal of Natural Medicines Volume: 72 Issue: 1 Pages: 32-42
  • DOI: 10.1007/s11418-017-1144-z
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A monoclonal antibody-based enzyme-linked immunosorbent assay for determination of homoharringtonine (2018)
  • Author(s): Pongkitwitoon, B., Sakamoto, S., Nagamitsu, R., Putalun W., Tanaka, H., Morimoto, S.
  • Journal Title: Planta Medica Volume: 印刷中 Issue: 14 Pages: 1038-1044
  • DOI: 10.1055/a-0578-8689
  • Peer Reviewed / Int'l Joint Research
• [Presentation] ピロリチジンアルカロイド、モノクロタリンの高感度免疫化学的測定法の確立 (2020)
  • Author(s): 坂元 政一、Gorawit Yusakul、長光 梨花、田中 宏幸、森元 聡
  • Organizer: 日本薬学会 第140年会
• [Presentation] Antiproliferative activity of sodium periodate-mediated harringtonine derivatives against HL-60 acute leukemia cells (2018)
  • Author(s): S. Sakamoto, T. Miyamoto, K. Usui, H. Tanaka, S. Morimoto
  • Organizer: The 5th International Conference on Current Drug Development 2018 (CDD 2018) and The 3rd International Conference on Herbal and Traditional Medicine 2018 (HTM 2018)
  • Int'l Joint Research
• [Presentation] Development of immunoassay for analysis of homoharringtonine in plant samples (2018)
  • Author(s): B. Pongkitwitoon, S. Sakamoto, R. Nagamitsu, W. Putalun, H. Tanaka, S. Morimoto
  • Organizer: 22nd International Phytopharm Congress 2018
  • Int'l Joint Research
• [Presentation] 植物二次代謝産物の免疫化学的分析法の開発 (2018)
  • Author(s): 坂元政一
  • Organizer: 日本生薬学会 第65回年会
• [Presentation] 過ヨウ素酸ナトリウムによるハリントニン誘導体の作出及びヒト前骨髄性白血病細胞HL60に対する増殖阻害活性 (2018)
  • Author(s): 坂元 政一、宮本 智文、臼井 一晃、田中 宏幸、森元 聡
  • Organizer: 日本薬学会 第138年会、石川県立音楽堂他(金沢)
• [Presentation] Cephalotaxus属由来ハリントニンの一本鎖抗体の作製及びその応用研究 (2017)
  • Author(s): 小松 頌平、坂元 政一、Gorawit Yusakul、Putalun Waraporn、田中 宏幸、森元 聡
  • Organizer: 日本生薬学会 第64回年会、東邦大学 習志野キャンパス(千葉)
• [Presentation] Immunoassays for cephalotaxus alkaloids, harringtonine and antiproliferative activity of its derivatives (2017)
  • Author(s): Sakamoto, S.
  • Organizer: 5th International Conference on Pharmaceuticals, Nutraceuticals and Cosmetic Science (IPNaCS 2017), The Tawana Hotel, Bangkok, Thailand
  • Int'l Joint Research / Invited