An improved method for the estimation of relative binding constants of t4 and its analogues with serum proteins.

A new method is reported for the determination of the binding constants of homologous ligands relative to that of the parent ligand. The technique minimizes the effects of random and bias errors inherent in absolute methods and obviates the need to quantitate the radiolabelled tracer concentration. In addition, it is likely to be of general applicability for monovalent proteins. The method has been applied in the determination of the binding constant (relative to that of T4) of the T4-analogue tracer used in the Amerlex free T4 radioimmunoassay, in respect of its binding to both T4 binding globulin and prealbumin. The binding constants of the analogue (relative to T4) were approximately 3% for both proteins. These levels were judged unlikely to interfere significantly with the measurement of free T4 in serum samples from patients with very wide variations in concentration of either T4 binding globulin or prealbumin.