Probing adsorbed fibronectin layer structure by kinetic analysis of monoclonal antibody binding

Adsorbed protein layers are often away from equilibrium and thus exhibit history dependent structures. We use the kinetics of monoclonal antibody binding, as measured using optical waveguide lightmode spectroscopy (OWLS), to investigate the structure of adsorbed fibronectin (Fn) layers formed under different kinetic paths. For all of the layers investigated, we find no difference between the apparent adsorption rate constants of (i) monoclonal antibodies specific to Fn's cell binding site (α-Fn) and (ii) monoclonal antibodies specific to cytochrome c (α-CC, as a control), indicating initial adsorption of antibodies to be non-specific. For certain layers, the saturation density and the initial projected area per antibody differ significantly between α-Fn and α-CC, suggesting specific binding to follow the initial non-specific attachment. The fraction of antibodies binding specifically to the Fn layer, and the number of Fn binding sites per specific binding event, are estimated in terms of the difference in initial projected areas between α-Fn and α-CC. For a Fn layer formed at a bulk concentration of 2 μg/mL, we find a decrease in specific binding with an increase in Fn layer formation time, suggesting post-adsorption structural changes of a lower density adsorbed layer diminish binding site availability. Conversely, for a Fn layer formed at a bulk concentration of 40 μg/mL, we find an increase in specific binding with an increase in the aging time of the Fn layer, implying post-adsorption structural changes reveal binding sites for a higher density adsorbed layer.