A1 Refereed original research article in a scientific journal
Controlled labelling of tracer antibodies for time-resolved fluorescence-based immunoassays
Authors: Kushnarova-Vakal, Anastasiia; Aalto, Rami; Huovinen, Tuomas; Wittfooth, Saara; Lamminmäki, Urpo
Publisher: Nature Publishing Group
Publication year: 2024
Journal: Scientific Reports
Journal name in source: Scientific reports
Journal acronym: Sci Rep
Article number: 18113
Volume: 14
Issue: 1
ISSN: 2045-2322
eISSN: 2045-2322
DOI: https://doi.org/10.1038/s41598-024-69294-7(external)
Web address : https://doi.org/10.1038/s41598-024-69294-7(external)
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/457433940(external)
Tracer antibodies, which are labelled with fluorescent or other type of reporter molecules, are widely employed in diagnostic immunoassays. Time-resolved fluorescence immunoassay (TRFIA), recognized as one of the most sensitive immunoassay techniques, utilizes tracers labelled with lanthanide ion (Ln) chelates. The conventional approach for conjugating isothiocyanate (ITC) Ln-chelates to antibodies involves random chemical targeting of the primary amino group of Lys residues, requiring typically overnight exposure to an elevated pH of 9-9.3 and leading to heterogeneity. Moreover, efforts to enhance the sensitivity of the assays by introducing a higher number of Ln-chelates per tracer antibody are associated with an elevated risk of targeting critical amino acid residues in the binding site, compromising the binding properties of the antibody. Herein, we report a method to precisely label recombinant antibodies with a defined number of Ln-chelates in a well-controlled manner by employing the SpyTag/SpyCatcher protein ligation technology. We demonstrate the functionality of the method with a full-length recombinant antibody (IgG) as well as an antibody fragment by producing site-specifically labelled antibodies for TRFIA for cardiac troponin I (cTnI) detection with a significant improvement in assay sensitivity compared to that with conventionally labelled tracer antibodies. Overall, our data clearly illustrates the benefits of the site-specific labelling strategy for generating high-performing tracer antibodies for TRF immunoassays.
Downloadable publication This is an electronic reprint of the original article. |  |
Funding information in the publication:
This research was supported by Business Finland (New Modalities Ecosystem Project, 448/31/2018) and Research Council of Finland’s Flagship InFLAMES (The funding decision numbers are 337530 and 357910). We would like to acknowledge the University of Turku Graduate School (UTUGS) and Turku University Foundation for supporting the doctoral researcher A.K.-V. We also thank to Jaana Rosenberg for the scientific discussions and technical assistance in HPLC experiments.
