Bioluminescent Antibodies through Photoconjugation of Protein G–Luciferase Fusion Proteins
- Simone F. A. WoutersSimone F. A. WoutersLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Den Dolech 2, 5612 AZ Eindhoven, The NetherlandsMore by Simone F. A. Wouters
- Willem J. P. VugsWillem J. P. VugsLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Den Dolech 2, 5612 AZ Eindhoven, The NetherlandsMore by Willem J. P. Vugs
- Remco ArtsRemco ArtsLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Den Dolech 2, 5612 AZ Eindhoven, The NetherlandsMore by Remco Arts
- Nynke M. de LeeuwNynke M. de LeeuwLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Den Dolech 2, 5612 AZ Eindhoven, The NetherlandsMore by Nynke M. de Leeuw
- Roy W. H. TeeuwenRoy W. H. TeeuwenLaboratory of Chemical Biology and Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Den Dolech 2, 5612 AZ Eindhoven, The NetherlandsMore by Roy W. H. Teeuwen
- Maarten Merkx*Maarten Merkx*Email: m.[email protected].Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Den Dolech 2, 5612 AZ Eindhoven, The NetherlandsMore by Maarten Merkx
Abstract

Bioluminescent antibodies represent attractive detection agents in both bioanalytical assays and imaging. Currently, their preparation relies on genetic fusion of luciferases to antibodies or nonspecific chemical conjugation strategies. Here, we report a generic method to generate well-defined covalent antibody–luciferase conjugates starting from commercially available monoclonal antibodies. Our approach uses fusion proteins consisting of the bright blue light-emitting luciferase NanoLuc (NL) and an Fc-binding protein domain (Gx) that can be photo-cross-linked to the antibody using UV light illumination. Green and red color variants were constructed by tight fusion of the NanoLuc with a green fluorescent acceptor domain and introduction of Cy3, respectively. To increase the already bright NanoLuc emission, tandem fusions were successfully developed in which the Gx domain is fused to two or three copies of the NanoLuc domain. The Gx-NL fusion proteins can be efficiently photo-cross-linked to all human immunoglobulin G (IgG) isotypes and most mammalian IgG’s using 365 nm light, yielding antibodies with either one or two luciferase domains. The bioluminescent antibodies were successfully used in cell immunostaining and bioanalytical assays such as enzyme-linked immunosorbent assay (ELISA) and Western blotting.





