Design of Polarity-Dependent Immunosensors Based on the Structural Analysis of Engineered Antibodies

Jiaul Islam, Paul Conroy, Christian Fercher, Mijin Kim, Zvi Yaari, Martina Jones, Toby D.M. Bell, Tom Caradoc-Davies, Ruby Law, James Whisstock, Daniel Heller, Stephen Mahler, Simon Corrie*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

“Reagentless” immunosensors are emerging to address the challenge of practical and sensitive detection of important biomarkers in real biological samples without the need for multistep assays and user intervention, with applications ranging from research tools to point-of-care diagnostics. Selective target binding to an affinity reagent is detected and reported in one step without the need for washing or additional reporters. In this study, we used a structure-guided approach to identify a mutation site in an antibody fragment for the polarity-dependent fluorophore, Anap, such that upon binding of the protein target cardiac troponin I, the Anap-labeled antibody would produce a detectable and dose-dependent shift in emission wavelength. We observed a significant emission wavelength shift of the Anap-labeled anti-cTnI mutant, with a blue shift of up to 37 nm, upon binding to the cTnI protein. Key differences in the resulting emission spectra between target peptides in comparison to whole proteins were also found; however, the affinity and binding characteristics remained unaffected when compared to the wild-type antibody. We also highlighted the potential flexibility of the approach by incorporating a near-infrared dye, IRDye800CW, into the same mutation site, which also resulted in a dose-dependent wavelength shift upon target incubation. These reagents can be used in experiments and devices to create simpler and more efficient biosensors across a range of research, medical laboratory, and point-of-care platforms.

Original languageAmerican English
Pages (from-to)1863-1871
Number of pages9
JournalACS Chemical Biology
Volume18
Issue number8
DOIs
StatePublished - 18 Aug 2023

Bibliographical note

Publisher Copyright:
© 2023 American Chemical Society.

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