2022 Fiscal Year Research-status Report
Development of a generalizable toolkit for rapidly improving the sensitivity of fluorescent immunosensor proteins
| Project/Area Number |
21K14468
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
朱 博 東京工業大学, 科学技術創成研究院, 助教 (70886605)
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | Immunosensor / mRNA display / Quenchbody / SARS-CoV-2 / Phage display / NGS |
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| Outline of Annual Research Achievements |
The research project is about making a toolkit for improving the performance of Quenchbody-based immunosensor (Quenchbody) in a short period. It will increase the feasibility of the quenchbody while responding to the needs of society in a fast manner, such as combating pandemics.
In the second year, the mRNA-display procedures were extensively optimized to get a functional and large Quenchbody for the linker screening. The cell-free synthesized Bone Gla protein (BGP) antibody fragment was successfully labeled with a fluorophore in an mRNA-display-required working procedure. The affinity and immunosensor function of the labeled antibody were confirmed. In addition, a phage-display-based Quenchbody library was created with unexpectedly good feasibility for functional linker sequence enrichment. Two rounds of selection of a BGP Quenchbody library with the randomized fluorophore linker sequences were performed and a clear motif was enriched, which was confirmed via next-generation sequencing.
In the meanwhile, the crowding agent function for rapidly improving the Quenchbody function was fully tested on a SARS-CoV-2 Quenchbody. The related results have been published in an International journal and the paper was selected as HOT Article and a Back Cover.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The research progress was slightly affected by the COVID-19 pandemic. An alternative phage display-based Quenchbody library was successfully created with unexpectedly better feasibility to screen linker sequences. Therefore, additional experiment is needed to finalize the rest of the screening and analysis for a higher grade outcome.
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| Strategy for Future Research Activity |
The mRNA-display procedures for Quenchbody will be further optimized for producing a useful Quenchbody library. The alternative and robust Quenchbody-displayed phage library will be screened to find better linker sequences for improved response and signal amplification. One of the targeting antibodies will be shifted from the cortisol antibody to another biomarker thyroxine with better affinity. Instead of the linear model, a machine learning-based model will be generated to predict the function of the linkers by using the next-generation sequence datasets. The related output will be organized as another research article for publication and conference presentation.
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| Causes of Carryover |
The usage of the mRNA crosslinking reagent, cell-free protein synthesis kits, and capturing antibodies is reduced due to the slightly delayed progress. And this part of the budget will be mainly used for additional experiments and paper publication mentioned above as planned.
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