Fluorescence labeling-induced structural rearrangement of a monoclonal IgG revealed by biophysical experiments and...

Although it is known that labeling antibodies with fluorescent dyes impairs their function, the underlying mechanism remains unclear. In this study, we show that multiple antibody functions decline in a strikingly similar way as the degree of labeling increases, suggesting that labeling induces a global structural change affecting the entire IgG molecule. Fluorescence anisotropy decay experiments revealed faster nanosecond-scale dynamics in labeled antibodies. FRET measurements showed that the Fc region moves closer to the hypervariable region upon labeling, further indicating major structural rearrangements. Molecular dynamics simulations confirmed that labeled antibodies adopt a more compact structure, bringing the Fc and Fab regions closer together. During simulations, the dye molecules gradually became less exposed to solvent, implicating the hydrophobic effect as a driver of structural collapse. The reduced antigen-binding affinity caused by labeling was partially restored by glycerol, likely due to its ability to weaken hydrophobic interactions. Together, these findings provide molecular-level insight into how fluorescent labeling disrupts antibody structure and function.