Optimised nanobody-based quenchbodies for enhanced protein detection
Quenchbodies, antibodies labelled with fluorophores that increase in intensity upon antigen binding, offer great promise for biosensor development. Nanobody-based quenchbodies are particularly attractive due to their small size, ease of expression, high stability, rapid evolvability, and amenability to protein engineering. However, existing designs for protein detection show limited dynamic range, with fluorescence increases of only 1.1-1.4 fold. Here we identify the tryptophan residues in the nanobody complementarity-determining regions (CDRs) that are critical to quenchbody performance. Using a combination of rational design and molecular dynamics simulations, we developed an optimised nanobody scaffold with tryptophans introduced at key positions. We used this scaffold in an in vitro directed-evolution screen against human inflammatory cytokine interleukin-6 (IL-6). This yielded quenchbodies with 1.5-2.4-fold fluorescence increases, enabling IL-6 detection down to 1-2 nM. Our scaffold provides a valuable platform for developing biosensors for diverse protein targets, with applications in research, diagnostics, and environmental monitoring.
Authors
- Andrew D. Griffiths (ORCID: https://orcid.org/0000-0002-0808-3539)
- Sebastian Hutchinson (ORCID: https://orcid.org/0000-0003-3895-0282)
- Haibo B. Yu (ORCID: https://orcid.org/0000-0002-1099-2803)
- Harshad Ghodke (ORCID: https://orcid.org/0000-0002-6628-876X)
- Richard R. Spinks (ORCID: https://orcid.org/0000-0002-8485-2336)
- Nirukshan Shanmugam (ORCID: https://orcid.org/0000-0002-8575-8640)
- Nehad El Salamouni (ORCID: https://orcid.org/0000-0001-6856-6332)
- Lisanne M. Spenkelink (ORCID: https://orcid.org/0000-0002-5511-8757)
- Adeline Pichard-Kostuch (ORCID: https://orcid.org/0000-0001-9220-8535)
- Marco Ribezzi‐Crivellari (ORCID: https://orcid.org/0000-0003-1217-5572)
- Conall Mc Guinness (ORCID: https://orcid.org/0000-0002-9733-6699)
- Jordan H. Cater (ORCID: https://orcid.org/0000-0003-3608-929X)
- Ghada H. Mansour (ORCID: https://orcid.org/0000-0001-8316-2619)
- Antoine M. van Oijen (ORCID: https://orcid.org/0000-0002-1794-5161)
- Stefan H. Mueller (ORCID: https://orcid.org/0000-0002-0930-2000)
- Viktor Zahoransky
Institutions
- University of Wollongong (AU)
- Université Paris Sciences et Lettres (FR)
- ESPCI Paris (FR)
Publication Details
- Journal
- Communications Biology
- Published
- 2025-06-18
- DOI
- https://doi.org/10.1038/s42003-025-08359-3
- Citations
- 7
- Primary Topic
- Monoclonal and Polyclonal Antibodies Research
- Type
- article
- Field-Weighted Citation Impact
- 9.52
Funders
- National Computational Infrastructure
- Medical Research Council
- National Health and Medical Research Council
- National Cancer Institute