Study of binding and denaturation dynamics of IgG and anti-IgG using dual color fluorescence correlation spectroscopy

In this article, we present a systematic study on IgG and Fab fragment of anti-IgG molecules using fluorescence auto- and cross-correlation spectroscopy to investigate their diffusion characteristics, binding kinetics, and the effect of small organic molecule, urea on their binding. Through our analysis, we found that the diffusion coefficient for IgG and Fab fragment of anti-IgG molecules were 37 ± 2 μm² s^-1 and 56 ± 2 μm² s^-1, respectively. From the binding kinetics study, the respective forward (k_a) and backward (k_d) reaction rates were (5.25 ± 0.25) × 10⁶ M^-1 s^-1 and 0.08 ± 0.005 s^-1, respectively and the corresponding dissociation binding constant (K_D) was 15 ± 2 nM. We also found that urea inhibits the binding of these molecules at 4 M concentration due to denaturation.