プレプリント / バージョン1

Detection of a Large Antigen through the Masking and Exposure of Fragment of Split Luciferase

##article.authors##

  • Cheng Qian Graduate School of Life Science and Technology, Institute of Science Tokyo
  • Ayumu Ninomiya Graduate School of Life Science and Technology, Institute of Science Tokyo
  • Natsuki Shibukawa Graduate School of Life Science and Technology, Institute of Science Tokyo
  • Hiroshi Ueda Laboratory for Chemistry and Life Science, Institute of Integrated Research, Institute of Science Tokyo
  • Takanobu Yasuda Laboratory for Chemistry and Life Science, Institute of Integrated Research, Institute of Science Tokyo
  • Bo Zhu Laboratory for Chemistry and Life Science, Institute of Integrated Research, Institute of Science Tokyo
  • Kitaguchi, Tetsuya Laboratory for Chemistry and Life Science, Institute of Integrated Research, Institute of Science Tokyo

DOI:

https://doi.org/10.51094/jxiv.967

キーワード:

protein M、 split luciferase、 NanoLuc、 homogeneous immunoassay

抄録

We developed PMBiT, an antibody-binding Protein M (PM)-based bioluminescent probe, which detects large antigens through luciferase reconstitution by exposing the luciferase fragment. The detection is achieved by exploiting the principle that the antibody, the large antigen, and PM are not able to form a complex at the same time. The PMBiT is prepared by conjugating PM with HiBiT-based peptide from the split NanoLuc luciferase through click reaction. It retained its binding activity to antibody, and showed bioluminescence upon reconstitution of the luciferase, by assembling with LgBiT, the other fragment of split NanoLuc. Mixing PMBiT with various IgG antibodies resulted in decreased bioluminescence. In contrast, when PMBiT was mixed with IgG bound to its large antigen, such as human C-reactive protein, the decreased bioluminescence was lessened, leading to bioluminescence increase in a dose dependent manner. Molecular dynamics simulations of PM showed that two regions in the C-terminus contribute to steric clashes with antigens due to their relatively rigid structures. Furthermore, in silico analysis of the structure suggested that antigen size is the primary factor blocking PMBiT binding to IgG for antigen detection. The immunoassay utilizing PMBiT does not require genetic manipulation of antibodies, allowing for seamless and scalable antibody replacement, and will advance the future of on-site detection and rapid diagnostics.

利益相反に関する開示

H.U., T.Y., B.Z., and T.K. received honoraria from HikariQ Health Inc. for an unrelated project.

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公開済


投稿日時: 2024-11-21 04:15:20 UTC

公開日時: 2024-11-21 08:59:26 UTC
研究分野
生物学・生命科学・基礎医学