Effect of surface packing density of interfacially adsorbed monoclonal antibody on the binding of hormonal antigen human chorionic gonadotrophin.
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.
Biological Physics Group, School of Physics and Astronomy, University of Manchester, Sackville Street Building, Sackville Street, Manchester M60 1QD, United Kingdom.
Hai
H
Xu
Jian R
JR
Lu
David E
DE
Williams
D000327:Adsorption; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D012822:Silicon Dioxide / Q000737:chemistry; D012996:Solutions / Q000737:chemistry; D013057:Spectrum Analysis; D013499:Surface Properties
D000911:Antibodies, Monoclonal; D006063:Chorionic Gonadotropin; D012996:Solutions; D012822:Silicon Dioxide