Double-fluorescent-labeled single-chain antibodies showing antigen-dependent fluorescence ratio change

Fluorescence ratio probes are useful tools for quantitative detection of target molecules even if the concentration of probe molecules is unknown. However, a general and widely applicable method for design and synthesis of fluorescence ratio probes has never been established. Here, we developed double-labeled single-chain antibody fragment (scFv) derivatives showing antigen-dependent fluorescence ratio change based on fluorescence resonance energy transfer (FRET) and antigen-dependent fluorescence quenching. Double-labeled scFvs were synthesized by incorporating fluorescent nonnatural amino acids labeled with TAMRA and Rhodamine Green (RhG) as a FRET donor and acceptor pair into N- and C-termini of scFv, respectively, using four-base and amber codons in a cell-free translation system. The resulting double-labeled antibody fragments showed significant fluorescence ratio change upon the antigen-binding. This result was explained by FRET occurring from RhG to TAMRA but TAMRA being quenched by Trp residues in the absence of antigen. The binding of the antigen canceled the quenching of TAMRA without changing FRET efficiency. Double-labeled antibody fragments developed here will be useful as diagnostic and imaging tools.