(575e) Antibody Adsorption and Orientation On Hydrophobic Surfaces

The orientation of a monoclonal IgG1 antibody on a model hydrophobic surface was studied by monitoring film rigidity and affinity for probe molecules as a function of surface coverage. Adsorption of the antibody onto an alkanethiol self-assembled monolayer on gold was monitored by quartz crystal microbalance with dissipation (QCM-D), which provides information on the viscoelastic properties of the layer in addition to adsorbed mass. The binding of probe molecules with affinity for the Fab region (the antigen) and Fc region (bacteria-derived protein G') were measured at different antibody surface densities after a bovine serum albumin blocking step. Based on an analysis of the film rigidity and binding patterns as a function of antibody surface coverage, it was found that the orientation of the antibody is not uniform throughout the creation of the film. At surface coverage below half of a monolayer, antibodies were found to adsorb side-on: rigidly coupled to the surface and unable to capture their antigen, while the Fc region was able to bind protein G'. Above half of a monolayer, antibodies adsorbed with more vertical orientations: more loosely coupled to the surface and able to bind more antigen and protein G' per molecule than at lower coverage. Beyond monolayer coverage, additionally adsorbing antibodies formed a diffuse layer that could be penetrated by protein G' and was even less rigidly coupled to the surface. This study shows that antibody films can be quite inhomogeneous and that the calculated affinity constant for an antibody-antigen interaction can depend on the antibody surface density at which it is measured.