INVESTIGATION OF IMMUNE-REACTIONS IN A FLOW-INJECTION SYSTEM USING SURFACE-PLASMON RESONANCE

The effect of flow rate on the binding of polyclonal sheep immunoglobulin G (IgG) and anti-sheep IgG to gold, and to immobilized anti-IgG and IgG receptor layers, respectively, has been studied using the optical technique of surface plasmon resonance at a gold-solution interface. The kinetics of protein binding were investigated by monitoring the reflectivity of light at a fixed angle close to the plasmon resonance. Fixed-angle reflectivity data are presented in terms of protein surface coverage. IgG and anti-IgG effective thicknesses were determined by fitting the full angle-dependent reflectivity data to Fresnel theory. The effective thicknesses of IgG and anti-IgC bound to the gold surface and to each other (as pre-adsorbed layers) were found to be independent of the flow rate of protein solution. Gold-adsorbed IgG and anti-IgG layers were found to be 63 +/- 15 Angstrom and 68 +/- 10 Angstrom thick, respectively, and 91 +/- 11 Angstrom (anti-IgG) and 20 +/- 7 Angstrom (IgG) thick when subsequently bound to their biospecific partner. The differences in the thickness values between the gold-adsorbed and protein-adsorbed layers are explained in terms of the orientation of the immobilized proteins and their deformation upon binding to the gold surface. Non-specific binding between immobilized sheep IgG layers and anti-mouse IgG was shown to be negligible.