Relationship between the structural conformation of monoclonal antibody layers and antigen binding capacity.
Neutron reflection has been used to determine the pH-dependent structural conformation of monoclonal antibody layers adsorbed at the hydrophilic silicon oxide/solution interface, within the pH range 4-8, over which the silicon oxide surface carried weak negative charges and the net charge on the antibody reversed. The depth resolution achieved, by use of D2O as solvent to enhance the neutron contrast of the adsorbed antibody layer, was around 2-3 A. The results have been correlated with the ellipsometric measurement of antigen binding capacity (AgBC). The antibody was a mouse monoclonal anti-hCG (human chorionic gonadotropin) directed against the beta subunit of hCG, with molecular weight of 150 000 and isoelectric point around pH 6.0. At pH 4, the adsorbed antibody could be described as a single layer 40 A thick, consistent with an almost perfect flat-on orientation with all three fragments (Fc, Fab) lying flat on the surface. With increasing pH, the antibody layer swelled (65 A at pH 6, 75 A at pH 8) and could be described as three sublayers of different protein density, consistent with some twisting of molecules so that some fragments became more loosely attached to the surface. At pH 8, the repulsive interaction between protein and surface was reflected in a significantly decreased total adsorbed amount. The dominant effect acting to increase AgBC was decreased surface packing density. The effect of the conformational changes revealed at different pH was less important. The results have shown that within the flat-on orientation adopted by the adsorbed antibody, steric hindrance is the main constraint on binding, restricting the access of the antigen to active sites within the antibody layer.
Xu
Hai
H
Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjing West Road, Qingdao Economic Development Zone, Qingdao 266555, China.
Zhao
Xiubo
X
Lu
Jian R
JR
Williams
David E
DE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000276:immunology; D001666:Binding Sites, Antibody; D006063:Chorionic Gonadotropin / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D051379:Mice; D009502:Neutrons; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000941:Antigens; D006063:Chorionic Gonadotropin