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-T4 Fab fragment were determined using a single high-density L-T4 biosensor surface in a BIAcore surface plasmon resonance instrument. The high-density L-T4 analog biosensor was calibrated by determination of the initial binding rate was of known concentrations of free anti-T4 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-T4 Fab fragment. The concentration of free anti-T4 Fab fragment in each solution at equilibrium was determined, and the equilibrium dissociation constant (KD) for each case was derived. The KD values determined in solution are compared to values determined by a direct kinetic analysis on the BIAcore instrument using individual biosensor surfaces.