Immunoassay reagents for thyroid testing.: 3.: Determination of the solution binding affinities of a T4 monoclonal antibody Fab fragment for a library of thyroxine analogs using surface plasmon resonance

A library of thyroxine analogs and tracers was prepared, and their solution binding affinities for an anti-T-4 Fab fragment were determined using a single high-density L-T-4 biosensor surface ina BIAcore surface plasmon resonance instrument. The high-density L-T-4 analog biosensor was calibrated by determination of the initial binding rate was of known concentrations of free anti-T-4 Fab fragment in solution to the biosensor surface. A range of individual thyroxine analog and tracer concentrations was subsequently mixed with a fixed concentration of anti-T-4 Fab fragment. The concentration of free anti-T-4 Fab fragment in each solution at equilibrium was determined, and the equilibrium dissociation constant (K-D) for each case was derived. The K-D values determined in solution are compared to values determined by a direct kinetic analysis on the BIAcore instrument using individual biosensor surfaces.