Orientation of a monoclonal antibody adsorbed at the solid/solution interface: A combined study using atomic force microscopy and neutron reflectivity

Conformational orientations of a mouse monoclonal antibody to the β unit of human chorionic gonadotrophin (anti-β-hCG) at the hydrophilic silicon oxide/water interface were investigated using atomic force microscopy (AFM) and neutron reflectivity (NR). The surface structural characterization was conducted with the antibody concentration in solution ranging from 2 to 50 mg·L-1 with the ionic strength kept at 20 mM and pH = 7.0. It was found that the antibody adopted a predominantly "flat-on" orientation, with the Fc and two Fab fragments lying flat on the surface. The AFM measurement revealed a thickness of 30-33 Å of the layer formed in contact with 2 mg·L-1 antibody in water, but, interestingly, the flat-on antibody molecules formed small nonuniform clusters equivalent to 2-15 antibody molecules. Parallel AFM scanning in air revealed even larger surface clusters, suggesting that surface drying induced further aggregation. The AFM study thus demonstrated that the interaction between protein and the hydrophilic surface is weak and indicated that surface aggregation can be driven by the attraction between neighboring protein molecules. NR measurements at the solid/water interface confirmed the flat-on layer orientation of adsorbed molecules over the entire concentration range studied. Thus, at 2 mg·L-1, the adsorbed antibody layer was well represented by a uniform layer with a thickness of 40 Å. This value is thicker than the 30-33 Å observed from AFM, suggesting possible layer compression caused by the tip tapping. An increase in the antibody concentration to 10 mg·L-1 led to increasing surface adsorption. The corresponding layer structure was well represented by a three-layer model consisting of an inner sublayer of 10 Å, a middle sublayer of 30 Å, and an outer sublayer of 25 Å, with the protein volume fractions in each sublayer being 0.22, 0.42, and 0.10, respectively. The structural transition can be interpreted as a twisting and tilting of segments of the adsorbed molecules, driven by an electrostatic repulsion between them that increases with the surface packing density. Hindrance of antigen access to antibody binding sites, resulting from the change in surface packing, can account for the decrease in antigen binding capacity (AgBC) with increasing surface density of the antibody that is observed. © 2006 American Chemical Society.