Preparation of Quenchbodies by protein transamination reaction.

Quenchbody (Q-body) is an antibody fragment labeled with fluorescent dye(s), which functions as a biosensor via the antigen-dependent removal of the quenching effect on fluorophores. It is based on the principle that the fluorescence of the dye(s) attached to the antibody N-terminal region is quenched primarily by the tryptophan residues present in the variable regions, and this quenching is released when the antigen binds to the antibody, resulting in increased fluorescence intensity. Hence Q-body is utilized in various immunoassays for the rapid and sensitive detection of analytes. So far, Q-bodies have been prepared by using a cell-free translation system or by combining Escherichia coli expression and post-labeling steps. However, the above methods need antibody gene cloning, and are time-consuming. In this study, we report a novel approach to prepare Q-bodies by protein N-terminal transamination. We used the antigen-binding fragment (Fab) of an antibody against the bone-Gla-protein (BGP), a biomarker for bone diseases, which was expressed in E. coli. The purified Fab was treated with Rapoport's salt to convert the amino group at the N-terminus to a ketone group, which in turn was allowed to react with fluorescent probes that have aminooxy or hydrazide groups, to prepare a Q-body. The Q-body prepared by this method could detect the BGP-C7 antigen at concentrations as low as 10 nM. Since the approach can label the protein N-terminus directly, it could be applied for preparing Q-bodies from natural antibodies and for the rapid screening of high-performance Q-bodies.