Strategy for Making a Superior Quenchbody to Proteins: Effect of the Fluorophore Position

Antibody-based sensors have made outstanding contributions to the fields of molecular biology and biotechnology. Our group recently developed a novel powerful fluorescent immunosensor strategy named Quenchbody (Q-body), which has been applied to the detection of a range of antigens in a rapid, simple, and sensitive manner. However, there were some Q-bodies whose fluorescence response was limited, especially for detecting protein antigens. With the aim of improving this issue, here we made twelve types of Q-bodies incorporated with different number and position of TAMRA fluorophore in the single chain Fv of HyHEL-10, an anti-hen egg lysozyme antibody, as a model. By measuring the fluorescence intensity and its antigen dependency, it was revealed that V-L-V-H type Q-bodies labeled at a non-CDR loop region of the V-L shows the highest fluorescence response. This position locates close to the quenching Trp35 in V-L, while it is far from Trp residues in the bound antigen. This result clearly suggests the importance of dye position to maximize the fluorescence quenching and antigen-dependent de-quenching. The discovery may open a way to make many other Q-bodies with superior response.