Application of surface plasmon resonance toward studies of low-molecular-weight antigen-antibody binding interactions.
Methods for studying low-molecular-weight antigen-antibody binding interactions using surface plasmon resonance detection are presented. The experimental parameters most relevant to studies of low-molecular-weight antigen-antibody binding interactions are discussed. Direct kinetic analysis of the binding interactions is most informative, providing both apparent association and dissociation rate constants from which equilibrium constants can be calculated. Equilibrium analysis, including steady-state and solution affinity studies, offers an alternative approach to direct kinetic analysis when knowledge of the individual kinetic rate constants is not required or difficult to determine. The various methods are illustrated by studies of an anti-T(4) Fab fragment binding interaction with several thyroxine analogs. The methods utilized were dependent on the affinity of the interaction. The high-affinity anti-T(4) Fab fragment/l-T(4) binding interaction was evaluated using direct kinetic analysis. An intermediate affinity anti-T(4) Fab fragment/l-T(3) binding interaction was evaluated using a combination of direct kinetic analysis, steady-state analysis, and solution affinity analysis. The relatively weak anti-T(4) Fab fragment/l-T(2) binding interaction was evaluated using steady-state and solution affinity analysis protocols. Several thyroxine 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.
Adamczyk
M
M
Diagnostics Division Organic Chemistry (9-NM), Abbott Laboratories, Building AP 20, 100 Abbott Park Road, Abbott Park, Illinois 60064, USA. maciej.adamczyk@abbot.com
Moore
J A
JA
Yu
Z
Z
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000937:Antigen-Antibody Reactions; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D008970:Molecular Weight; D020349:Surface Plasmon Resonance; D013974:Thyroxine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D013974:Thyroxine