Effect of antibody affinity on the isotherm of antibody binding to surface-immobilized antigen.
The binding of monoclonal antibodies to surface-adsorbed antigen was studied. Mouse IgG antibodies directed against dinitrophenyl groups (DNP) and O6-ethyl-2'-deoxyguanosine with known affinity for the antigen were used. The hapten was coupled to a protein, bovine serum albumin (BSA) or keyhole limpet hemocyanin, and adsorbed to polystyrene or silicone surfaces. Four different DNP-BSA epitope densities were used. Antibodies were incubated with the antigen-coated surface overnight. The bound antibodies were detected either optically by ellipsometry or by enzyme-conjugated anti-mouse IgG antibodies in the common ELISA technique. Absorbance values from ELISA measurements were transformed to surface density through calibration by ellipsometry. The experimental data showed that the binding of a high affinity antibody (Ka = 2.0 X 10(10] was diffusion rate limited after 24 h incubation time. Identical binding isotherms were found for high and low affinity clones of anti-DNP antibodies (Ka = 4.1 X 10(7) and 3.5 X 10(5] when antigen of high epitope density was used. At low epitope density the amount of bound low affinity antibodies decreased. Electron microscopy was used for studies of the distribution of colloidal gold-antibody complexes bound to surface-immobilized antigen. The results of the experiment showed that low affinity antibodies were bound in clusters whereas high affinity antibodies bound as single particles. These findings were related to the ELISA measurements. The results indicate that the binding isotherm of antibody to surface adsorbed antigen is not merely a reflection of the intrinsic antibody affinity measured in solution. Other macromolecular properties of antibodies, e.g., lateral intermolecular interactions and phase separation, affect the heterogeneous binding reaction.
Werthén
M
M
Department of Histology, University of Gothenburg, Sweden.
Nygren
H
H
D000818:Animals; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D004140:Dinitrophenols / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006046:Gold; D006241:Haptens; D046911:Macromolecular Substances; D051379:Mice; D008854:Microscopy, Electron; D012710:Serum Albumin, Bovine / Q000276:immunology*; D013499:Surface Properties; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D004140:Dinitrophenols; D000939:Epitopes; D006241:Haptens; D046911:Macromolecular Substances; C034638:dinitrophenyl-bovine serum albumin; D012710:Serum Albumin, Bovine; D006046:Gold