Orientation of a Monoclonal Antibody Adsorbed at the Solid/Solution Interface:  A Combined Study Using Atomic Force Microscopy and Neutron Reflectivity

Abstract

Conformational orientations of a mouse monoclonal antibody to the beta unit of human chorionic gonadotrophin (anti-beta-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 A 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 A. This value is thicker than the 30-33 A 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 A, a middle sublayer of 30 A, and an outer sublayer of 25 A, 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.

Interfacial recognition of human prostate-specific antigen by immobilized monoclonal antibody: effects of solution conditions and surface chemistry

“…The volume fraction of the middle layer was reduced slightly to 52.5 per cent, but its thickness was increased to 30 Å , showing that this layer was dominant and contained the majority of the antibody. The total surfaceadsorbed amount reached a maximum of 2.9 mg m -2 , which is consistent with the proximity to the pI of the antibody of around 5.5 [4]. A further increase in pH to 6.1 reduced both thickness and volume fraction of the middle layer, which is consistent with the decline of surface adsorption beyond the pI.…”

“…The solution was then replaced by a pH 4 buffer, followed by a pH 4 antibody solution. Desorption was observed, and the amount of the adsorbed antibody reduced to the previous level at pH 4, showing that the adsorption was entirely reproducible under the experimental conditions. These results are highly consistent with our previous investigations of anti-human chorionic gonadotropin (anti-hCG) systems, in which the solution pH was changed from 4 to 8, then brought back to 4 again [5].…”

Different Organization of Type I Collagen Immobilized on Silanized and Nonsilanized Titanium Surfaces Affects Fibroblast Adhesion and Fibronectin Secretion

“…10,11,19,23,25,28 Additional factors that could explain the smaller amount of collagen immobilized in the PL-GP surface are the lower silanization coverage eventually limiting the amount of collagen that can be adsorbed from higher concentration solutions and that the epoxy organofunctional group of GPTES is susceptible to hydrolyzation when exposed to acidic aqueous solutions in the long term. 56 Since the reaction between silanes and collagen is performed at a pH ≈ 6, it is possible that some GPTES molecules undergo hydrolysis, consequently hampering further collagen bonding.…”

Interfacial assembly of proteins and peptides: recent examples studied by neutron reflection

“…Interfacial assembly of proteins X. Zhao et al S663 (Xu et al 2006b). Figure 3a shows the best fits to the measured reflectivity in the presence of 2 mg l 21 of anti-b-hCG in D 2 O by assuming that the antibodies adopted 'flat-on' (solid line), 'side-on' (dashed line) and 'end-on' or 'head on' (dotted line) orientations (figure 3b).…”

“…Meanwhile, neither the best fits of 'side-on' nor 'end-on' orientations would fit the shape of the measured reflectivity profile. The adsorption of anti-b-hCG at higher solution concentrations was also studied and the best fits to the measured reflectivity profiles are shown in figure 4a (Xu et al 2006b). The D 2 O profile and the data at 2 mg l 21 are plotted together for comparison.…”