Real Time Analysis of Antibody‐Antigen Reaction Kinetics

Surface plasmon resonance, i.e. detection of changes in refractive index on a surface, was used in a biosensor to evaluate the dissociation/association rate and affinity constants of human monoclonal IgG and IgM antibodies and Fab fragments. The results showed that an observed difference in affinity constants between intact and fragmented IgG anti-tetanus antibody was related to approximately 10-fold differences in dissociation rate constants, since the association rate constants were in the same range, i.e. 2-3 x 10(5) (M-1 s-1). Affinity constants, as determined by conventional solid phase enzyme immunoassay, were substantially higher than the constants produced by the biosensor. Human monoclonal IgM anti-Tn alpha antibodies showed, furthermore, one order of magnitude higher association rate constants, as compared with the IgG antibodies, but since the dissociation rate constants were more than ten times higher, the resulting affinity constants of the anti-carbohydrate IgM antibodies were still somewhat lower than those of the IgG antibodies.