Force Probe Measurements of Antibody–Antigen Interactions

The surface force apparatus has been used to quantify directly the forces that govern the interactions between proteins and ligands. In this work, we describe the measured interactions between the antigen fluorescein and the Fabʹ fragment of the monoclonal 4-4-20 anti-fluorescyl IgG antibody. Here we first describe the use of the surface force apparatus to demonstrate directly the impact of the charge composition in the region of the antibody binding site on the antibody interactions. Several approaches are described for immobilizing antigens, antibodies, and proteins in general for direct force measurements. The measured force profiles presented are accompanied by an extensive discussion of protocols used to analyze the force–distance curves and to interpret them in terms of the antibody structure. In addition to long-range electrostatic forces, we also consider short-range forces that can affect the strength of adhesion between the Fabʹ and immobilized fluorescein. The latter investigations demonstrate the influence of interfacial properties on the recognition of surface-bound antigens.