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Bioorthogonal site-selective conjugation of fluorescent dyes to antibodies: method and potential applications


Grossenbacher, Philipp; Essers, Maria C; Moser, Joël; Singer, Simon A; Häusler, Stephanie; Stieger, Bruno; Rougier, Jean-Sébastien; Lochner, Martin (2022). Bioorthogonal site-selective conjugation of fluorescent dyes to antibodies: method and potential applications. RSC Advances, 12(44):28306-28317.

Abstract

Antibodies are immensely useful tools for biochemical research and have found application in numerous protein detection and purification methods. Moreover, monoclonal antibodies are increasingly utilised as therapeutics or, conjugated to active pharmaceutical ingredients, in targeted chemotherapy. Several reagents and protocols are reported to synthesise fluorescent antibodies for protein target detection and immunofluorescence applications. However, most of these protocols lead to non-selective conjugation, over-labelling or in the worst case antigen binding site modification. Here, we have used the antibody disulphide cleavage and re-bridging strategy to introduce bright fluorescent dyes without loss of the antibody function. The resulting fluorescent IgG1 type antibodies were shown to be effective imaging tools in western blot and direct immunofluorescence experiments.

Abstract

Antibodies are immensely useful tools for biochemical research and have found application in numerous protein detection and purification methods. Moreover, monoclonal antibodies are increasingly utilised as therapeutics or, conjugated to active pharmaceutical ingredients, in targeted chemotherapy. Several reagents and protocols are reported to synthesise fluorescent antibodies for protein target detection and immunofluorescence applications. However, most of these protocols lead to non-selective conjugation, over-labelling or in the worst case antigen binding site modification. Here, we have used the antibody disulphide cleavage and re-bridging strategy to introduce bright fluorescent dyes without loss of the antibody function. The resulting fluorescent IgG1 type antibodies were shown to be effective imaging tools in western blot and direct immunofluorescence experiments.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Clinic for Clinical Pharmacology and Toxicology
Dewey Decimal Classification:610 Medicine & health
Uncontrolled Keywords:General Chemical Engineering, General Chemistry
Language:English
Date:1 January 2022
Deposited On:04 Nov 2022 13:22
Last Modified:27 Jun 2024 01:40
Publisher:Royal Society of Chemistry
ISSN:2046-2069
OA Status:Gold
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1039/d2ra05580e
Project Information:
  • : FunderSNSF
  • : Grant ID206021_177033
  • : Project TitleA dual wavelength X-ray single crystal diffractometer for accurate investigations at extreme conditions
  • : FunderSNSF
  • : Grant IDCRSII5_180326
  • : Project TitleThe role of mitochondrial carriers in metabolic tuning and reprogramming by calcium flow across membrane contact sites
  • Content: Published Version
  • Language: English
  • Licence: Creative Commons: Attribution 3.0 Unported (CC BY 3.0)