Bioluminescent antibodies through photoconjugation of protein-G-luciferase fusion proteins

Bioluminescent antibodies represent attractive detection agents in both bioanalytical assays and imaging. Currently, their preparation relies genetic fusion of luciferases to antibodies or non-specific chemical conjugation strategies. Here we report a generic method to generate well-defined covalent antibody-luciferase conjugates starting from commercially available monoclonal anti-bodies. 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 photocrosslinked 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 2 or 3 copies of the NanoLuc domain. The Gx-NL fusion proteins can be efficiently photocrosslinked to all human IgG isotypes an...