Interleukin-2 monoclonal antibody affinity adsorption. The critical role of binding kinetics for optimal immunoadsorption.
With the ready availability of monoclonal antibodies reactive with an extensive spectrum of antigens, immunoaffinity adsorption has become more widely applicable for protein purification. However, given several monoclonal antibodies reactive with the same antigen, most investigators have found that only a few antibodies are useful for solid-phase immunoaffinity antigen purification. Accordingly, in order to determine the parameters of monoclonal antibody-antigen binding most important for effective immunoaffinity adsorption, equilibrium and kinetic binding experiments were performed using radiolabeled interleukin-2 (IL-2) as antigen and four different IL-2-reactive monoclonal antibodies. The antibodies were found to differ primarily with respect to their kinetic binding characteristics; at 37 degrees C IL-2 bound to two of these antibodies very rapidly, while it bound to the other two more slowly. When binding was performed at 4 degrees C, the equilibrium dissociation constants for all of the antibodies decreased due to a more marked prolongation of the dissociation rate than the association rate. However, at 4 degrees C the association rates of the two slow-reactive antibodies became retarded so markedly that efficient affinity adsorption did not occur. By comparison, for both of these antibodies, efficient removal of IL-2 could be obtained if adsorption was performed at 37 degrees C, provided the column flow rate was adjusted according to the IL-2 association rate. Kinetic considerations also dictated IL-2 adsorption to mixtures of two or more monoclonal antibodies: IL-2 immunoadsorption correlated with the association rates of the individual antibodies, rather than the equilibrium binding constants. These results indicate that the most important parameter for efficient affinity adsorption is the association rate constant. In addition, the results obtained indicate that monoclonal antibodies may differ markedly as regards their kinetic binding characteristics, and that all antibodies can serve as effective immunoadsorbents, provided their antigen binding characteristics are known.
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000032:analysis*; D002846:Chromatography, Affinity / Q000379:methods; D007164:Immunosorbents; D007376:Interleukin-2 / Q000276:immunology / Q000302:isolation & purification*; D007700:Kinetics; D013696:Temperature
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007164:Immunosorbents; D007376:Interleukin-2