Kinetic study of the adsorption of human serum albumin on immobilized antibody using the split-peak effect in immunochromatography.

The split-peak effect was used to determine the association rate constant of the antigen-immobilized antibody reaction. The amount of immobilized human serum albumin antibody on the chromatographic support was varied in order to find the optimal conditions to reduce the mass transfer contribution in the stagnant mobile phase fluid and measure the effective association rate constant of human serum albumin with the immobilized antibody. Kinetic studies as a function of flow-rate demonstrate the validity of the method consisting in determining the association rate constant from measurements performed on columns of various capacities. These experiments show that limitations due to mass transfer to the surface of the adsorbent are minimized at high flow-rates and for a low density of immobilized ligand.