Interaction of triiodothyronine-biotin conjugate with binding proteins in immunoassay systems

The study presents a new design of a test system for the detection of a thyroid hormone, free 3,3??,5-triiodo-L-thyronine (free T₃), by enzyme-linked immunosorbent assay (ELISA) and immunoradiometric assay (IRMA) in human blood serum. For this purpose, a low-molecular-weight bifunctional conjugate of T₃ with biotin (Bt) was synthesized. The conjugate T₃-Bt can be bound to biotin-binding proteins on a solid support via its biotin residue, and T₃ portion of the conjugate can interact with a monoclonal antibody against T₃ (anti-T₃-MAb) that is labeled with horseradish peroxidase or iodine-125 in ELISA and IRMA, respectively. The immunochemical interaction is hindered if the T₃ residue is involved in a preformed complex of T₃-Bt with avidin in a solid phase. Computer simulations revealed that the iodothyronine residue is shielded by a glycan component of avidin in such a complex. Binding of T₃-Bt to both T₃-free sites of anti-T₃-MAb and to avidin on a solid support was achieved by delayed introduction of T₃-Bt into avidin-coated plates that were preliminarily co-incubated with labeled anti-T₃-MAb and the analyzed T₃ sample.