Design of Polarity-Dependent Immunosensors Based on the Structural Analysis of Engineered Antibodies
“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...
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Published in | ACS chemical biology Vol. 18; no. 8; pp. 1863 - 1871 |
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Main Authors | , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
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United States
American Chemical Society
18.08.2023
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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. |
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AbstractList | "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. |
Author | Kim, Mijin Whisstock, James Islam, Jiaul Caradoc-Davies, Tom Mahler, Stephen Corrie, Simon Conroy, Paul Law, Ruby Bell, Toby D. M. Fercher, Christian Heller, Daniel Yaari, Zvi Jones, Martina |
AuthorAffiliation | Molecular Pharmacology Program Department of Chemical and Biological Engineering School of Pharmacy, Faculty of Medicine Australian Institute for Bioengineering and Nanotechnology (AIBN) ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology (AIBN) Dept. of Biochemistry and Molecular Biology, Biomedicine Discovery Institute School of Chemistry |
AuthorAffiliation_xml | – name: Australian Institute for Bioengineering and Nanotechnology (AIBN) – name: Molecular Pharmacology Program – name: School of Pharmacy, Faculty of Medicine – name: Department of Chemical and Biological Engineering – name: Dept. of Biochemistry and Molecular Biology, Biomedicine Discovery Institute – name: ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology (AIBN) – name: School of Chemistry |
Author_xml | – sequence: 1 givenname: Jiaul surname: Islam fullname: Islam, Jiaul organization: Department of Chemical and Biological Engineering – sequence: 2 givenname: Paul surname: Conroy fullname: Conroy, Paul organization: Dept. of Biochemistry and Molecular Biology, Biomedicine Discovery Institute – sequence: 3 givenname: Christian surname: Fercher fullname: Fercher, Christian organization: ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology (AIBN) – sequence: 4 givenname: Mijin orcidid: 0000-0002-7781-9466 surname: Kim fullname: Kim, Mijin organization: Molecular Pharmacology Program – sequence: 5 givenname: Zvi surname: Yaari fullname: Yaari, Zvi organization: School of Pharmacy, Faculty of Medicine – sequence: 6 givenname: Martina surname: Jones fullname: Jones, Martina organization: ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology (AIBN) – sequence: 7 givenname: Toby D. M. orcidid: 0000-0002-4570-5595 surname: Bell fullname: Bell, Toby D. M. organization: School of Chemistry – sequence: 8 givenname: Tom surname: Caradoc-Davies fullname: Caradoc-Davies, Tom organization: Dept. of Biochemistry and Molecular Biology, Biomedicine Discovery Institute – sequence: 9 givenname: Ruby surname: Law fullname: Law, Ruby organization: Dept. of Biochemistry and Molecular Biology, Biomedicine Discovery Institute – sequence: 10 givenname: James surname: Whisstock fullname: Whisstock, James organization: Dept. of Biochemistry and Molecular Biology, Biomedicine Discovery Institute – sequence: 11 givenname: Daniel orcidid: 0000-0002-6866-0000 surname: Heller fullname: Heller, Daniel organization: Molecular Pharmacology Program – sequence: 12 givenname: Stephen surname: Mahler fullname: Mahler, Stephen organization: Australian Institute for Bioengineering and Nanotechnology (AIBN) – sequence: 13 givenname: Simon orcidid: 0000-0001-8029-1896 surname: Corrie fullname: Corrie, Simon email: simon.corrie@monash.edu organization: Department of Chemical and Biological Engineering |
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Title | Design of Polarity-Dependent Immunosensors Based on the Structural Analysis of Engineered Antibodies |
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