Effect of antibody affinity on the isotherm of antibody binding to surface-immobilized antigen.

摘要: The binding of monoclonal antibodies to surface-adsorbed antigen was studied. Mouse IgG directed against dinitrophenyl groups (DNP) and O6-ethyl-2'-deoxyguanosine with known affinity for the were used. hapten coupled a protein, bovine serum albumin (BSA) or keyhole limpet hemocyanin, adsorbed polystyrene silicone surfaces. Four different DNP-BSA epitope densities Antibodies incubated antigen-coated surface overnight. bound detected either optically by ellipsometry enzyme-conjugated anti-mouse in common ELISA technique. Absorbance values from measurements transformed density through calibration ellipsometry. experimental data showed that high antibody (Ka = 2.0 X 10(10] diffusion rate limited after 24 h incubation time. Identical isotherms found low clones anti-DNP 4.1 10(7) 3.5 10(5] when At amount decreased. Electron microscopy used studies distribution colloidal gold-antibody complexes surface-immobilized antigen. results experiment clusters whereas as single particles. These findings related measurements. indicate isotherm is not merely reflection intrinsic measured solution. Other macromolecular properties antibodies, e.g., lateral intermolecular interactions phase separation, affect heterogeneous reaction.