Enzyme immunoassay for free thyroxin

We describe a mathematical model for a single-tube enzyme immunoassay for free thyroxin (FT4), involving use of a thyroxin/horseradish peroxidase (EC 1.11.1.7) conjugate that does not interact with thyroxin-binding globulin. In the presence of serum two populations of unassociated, or free, immunologically active constituents are present: FT4 and the conjugate. The concentrations of the former are determined by the serum constituents and of the latter by the albumin concentration. When a small quantity of antibody is added, it reacts with the variable amount of FT4 and with the constant amount of the conjugate, thus giving a measure of the FT4. We constructed a mathematical model based on thermodynamic binding constants and adsorption data. The model gives satisfactory agreement with the experimental data under a variety of experimental conditions. Results for 19 patients' serum samples demonstrate the validity of the concept.