Radioimmunoassay for Triiodothyronine (T3): I. Affinity and Specificity of the Antibody for T3*

Abstract

The free acid form of L-triiodothyronine was coupled to HSA, BSA or RSA using carbodiimide. The conjugates contained 11–17 moles of T₃ per mole of protein. Rabbits were immunized separately with each conjugate. Antibodies against T₃ were obtained in all animals after 6 weeks of immunization. One antiserum prepared with T₃-HSA was studied in detail using a radioimmunoassay procedure. It was useful in a final dilution of 1:1000 to 1:4000 and was sufficiently sensitive to measure 50 pg of unlabeled T₃ per assay tube. The ability of 30 tyrosine and thyronine derivatives to compete with T₃ for binding to antibody were investigated to assess the specificity of the assay. Antibody recognition involved the following structural requirements: two phenolic rings; a 3′ iodine; 4′ hydroxyl group and a 5′ hydrogen. Alterations in the alanine side chain reduced antibody recognition, but to a much smaller extent than changes in the structure of the phenolic rings. Thus, the antiserum is highly specific for L-T₃ and its alanine side chain derivatives. Preliminary results obtained with measurements of T₃ in human serum are given.