Development of a drug screening method using a recombinant antibody

• Project/Area Number: 16K09211
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Legal medicine
• Research Institution: Kumamoto University
• Principal Investigator: Sasao Ako 熊本大学, 大学院生命科学研究部(医), 助教 (80284751)
• Research Collaborator: Morioka Hiroshi ; Ueda Hiroshi ; Yamaguchi Yoshihiro
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2018: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000); Fiscal Year 2017: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2016: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
• Keywords: 抗薬物抗体 / 組換え抗体 / 薬物スクリーニング / 法医中毒学 / 免疫学的検出 / 薬毒物スクリーニング / フルボキサミン / 一本鎖抗体

Outline of Final Research Achievements

In this study, we aimed to provide a drug screening method using a recombinant antibody which was suitable for forensic practices. We used an antibody against anti-depressant fluvoxamine (FLV) and prepared a highly purified antibody-binding fragment(Fab). The Fab was mixed with excess FLV, and crystals of FLV-Fab complex were grown using hanging drop vapor diffusion technique. The crystallographic analysis will be invaluable in designing more potent antibodies in near future. We also created a FLV detection system using a Quenchbody (Q-body), a novel fluoroimmunosensor that can detect an antigen immediately without additional reagents or washing steps. We showed that the system could be applied to detect serum FLV with high sensitivity. This study demonstrates the potential of Q-body probes as a tool towards developing novel immunoassay applications for forensic practices.

Academic Significance and Societal Importance of the Research Achievements

法医学が担う実務において、その死因に薬毒物が関与していたかどうかを検査する事は重要である。現在、抗体を使った薬物検査法はよく利用されているが、検査できる薬物が限定的であり、類似化合物に対して擬陽性が起こる事など問題点も多い。本課題では、国内で中毒事例の多い向精神薬の一つであるFLVについて、薬物と抗体の相互作用部位を構造解析によって明らかにする事を試みた。本研究成果である結晶を用いた構造解析の知見は、異なる薬物に対する抗体作製への足掛かりとなるものである。また、本成果である作製したQuenchbodyは、異なる薬物に対する抗体遺伝子を挿入する事で、同様の検出法を直ちに提供できるツールとなる。

Research Products

• [Int'l Joint Research] Hongik University(韓国)
• [Journal Article] Development of a fluvoxamine detection system using a Quenchbody, a novel fluorescent biosensor. (2019)
  • Author(s): Sasao, A., Takaki, M., Jeong, H. J., Yonemitsu, K., Ohtsu, Y., Tsutsumi, H., Furukawa, S., Morioka, H., Ueda, H. and Nishitani, Y.
  • Journal Title: Drug Test Anal. Volume: 11 Issue: 4 Pages: 601-609
  • DOI: 10.1002/dta.2520
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Development of immunoassay for fluvoxamine detection using recombinant single-chain variable fragment antibody (2017)
  • Author(s): Ako Sasao, Michiyo Takaki, Yuki Ohtsu, Satoko Mishima, Kosei Yonemitsu, Hiroshi Morioka, Yoko Nishitani
  • Journal Title: Forensic Toxicology Volume: 印刷中 Issue: 2 Pages: 301-308
  • DOI: 10.1007/s11419-017-0358-9
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] 抗薬物抗体の抗原認識機構の解明を目指した 薬物-抗体タンパク質の共結晶作製 (2019)
  • Author(s): 笹尾亜子、黒澤凜、山口佳宏、大津由紀、 堤博志、古川翔太、米満孝聖、西谷陽子
  • Organizer: 第103次日本法医学会学術全国集会
• [Presentation] Development of a fluvoxamine detection system using Quenchbody, a novel antibody-based fluorescent biosensor (2018)
  • Author(s): Ako Sasao, Michiyo Takaki, Kyoko Hirata, Yuki Ohtsu, Hiroshi Tsutsumi, Shota Furukawa, Kosei Yonemitsu, Hiroshi Morioka, Hiroshi Ueda, Yoko Nishitani
  • Organizer: 24th Congress of the International Academy of Legal Medicine
  • Int'l Joint Research
• [Presentation] 抗うつ薬フルボキサミンの一本鎖抗体を用いたELISA定量法とその特性 (2017)
  • Author(s): 笹尾亜子, 髙木美智代, 大津由紀, 三島聡子, 堤 博志, 古川翔太, 米満孝聖, 森岡弘志, 西谷陽子
  • Organizer: 第101次日本法医学会学術全国集会
• [Presentation] 抗うつ薬フルボキサミンに対する蛍光免疫測定素子Quenchbodyの作製 (2016)
  • Author(s): 笹尾亜子, 髙木美智代, Hee-Jin Jeong, 大津由紀, 三島聡子, 米満孝聖, 上田 宏, 西谷陽子
  • Organizer: 第100次日本法医学会学術全国集会
  • Place of Presentation: 東京都
  • Year and Date: 2016-06-15