Effect of Surface Packing Density of Interfacially Adsorbed Monoclonal Antibody on the Binding of Hormonal Antigen Human Chorionic Gonadotrophin

Abstract

Interfacial adsorption of a mouse monoclonal antibody (type IgG1, anti-beta-hCG) at the hydrophilic silicon oxide/water interface has been studied by spectroscopic ellipsometry and neutron reflection, followed by assessment of binding of a hormonal antigen, human chorionic gonadotrophin (hCG), onto the adsorbed antibody molecules. The amount of adsorption reached a maximum around the isoelectric pH (IP) of 6 for the antibody; this pH-dependent pattern could be altered by increasing salt concentration, a trend also observed for other proteins. Neutron reflection revealed the formation of a 40 A uniform layer from the adsorbed antibody, indicating a flat-on orientation. The subsequent hCG binding showed that the molar ratio of hCG bound to antibody at the interface was as high as 0.7 at low surface coverage of antibody and decreased with increasing surface antibody concentration. The results point to an increasing extent of steric hindrance to hCG access with increasing packing density of antibody molecules on the surface. Comparison with previously published crystal structure studies suggests twisting of the variable region to allow access of the antigen. The binding of hCG was also found to be pH-dependent with its maximum around the IP, if the ionic strength of the solution was low (20 mM). However, if the ionic strength was increased to 200 mM, then hCG binding was influenced by a combination of steric hindrance and electrostatic interaction between the antigen and the surface. These results are highly relevant to the improvement of the performance of biotechnologies such as fertility test pads and biosensors based on antibody immobilization.

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

“…The determination of in situ conformational orientations of the antibody adsorbed at the solidsolution interface represents a major challenge for many existing technologies. Neutron reflection is by far the most reliable technique and has been used for determining the interfacial orientations of monoclonal antibodies (Xu et al 2006a(Xu et al ,b, 2007Lu et al 2007). …”

A Novel On‐Chip Method for Differential Extraction of Sperm in Forensic Cases

“…Further, this may point to a potential steric hindrance in higher mediator concentrations for SLeX immobilization. As reported in the literature, more densely packed layers revealed lower surface activity 32, 33. This effect also indicated the link between surface coverage, immobilization of molecules and capture activities, and therefore, a lower density of immobilization process on the surface provided a higher binding and sensitivity 32, 34.…”

“…As reported in the literature, more densely packed layers revealed lower surface activity 32, 33. This effect also indicated the link between surface coverage, immobilization of molecules and capture activities, and therefore, a lower density of immobilization process on the surface provided a higher binding and sensitivity 32, 34. Since 4‐ABAH has a role to immobilize SLeX molecules in the next step, much lower concentrations of 4‐ABAH will potentially not provide more binding points for binding of SLeX agent to the surface.…”

Engineering of PDMS surfaces for use in microsystems for capture and isolation of complex and biomedically important proteins: Epidermal growth factor receptor as a model system

“…25 Finally, the nature of attachment of antibody likely affects the antibody surface packing densities, which is known to affect the activity of the bound antibody. 26,27 Inspection of Figure 3 also reveals that a higher signal-to-background ratio was measured in these experiments with the PS capture surface (48:1 and 44:1 for H11 and 111.6, respectively) as compared to PDMS (21:1 and 14:1). We caution, however, that this result is influenced by the different scintillation counting methods used to acquire the data on PS as compared to PDMS.…”