Application of surface plasmon resonance toward studies of low-molecular-weight antigen-antibody binding interactions

Methods for studying low-molecular-weight antigen-antibody binding interact ions using surface plasmon resonance detection are presented. The experimen tal parameters most relevant to studies of low-molecular-weight antigen-ant ibody binding interactions are discussed. Direct kinetic analysis of the bi nding interactions is most informative, providing both apparent association and dissociation rate constants from which equilibrium constants can be ca lculated. Equilibrium analysis, including steady-state and solution affinit y studies, offers an alternative approach to direct kinetic analysis when k nowledge of the individual kinetic rate constants is not required or diffic ult to determine. The various methods are illustrated by studies of an anti -T-4 Fab fragment binding interaction with several thyroxine analogs. The m ethods utilized were dependent on the affinity of the interaction. The high -affinity anti-T-4 Fab fragment/L-T-4 binding interaction was evaluated usi ng direct kinetic analysis. An intermediate affinity anti-T-4 Fab fragment/ L-T-3 binding interaction was evaluated using a combination of direct kinet ic analysis, steady-state analysis, and solution affinity analysis. The rel atively weak anti-T-4 Fab fragment/L-T-3 binding interaction was evaluated using steady-state and solution affinity analysis protocols. Several thyrox ine tracers that could not be immobilized to a biosensor surface were also evaluated via the solution affinity format. In cases where a given binding interaction was examined using multiple methods the results were comparable . (C) 2000 Academic Press.