A New Principle of Thyroxine (T4) and Triiodothyronine (T3) Radioimmunoassay in Unextracted Serum Using Amisera with Binding Optima at Extreme pH Ranges1)

Summary: Thyroxine (T4) or triiodothyronine (T3) was covalently linked to bovine thyroglobulin (bTg) in a molar ratio of 25: l (T4:bTg) or 30:1 (T3:bTg) using a water-soluble carbodiimide. The conjugates of T4-bTg and T3-bTg were used for immunisation of rabbits. After 6 booster-injections all rabbits immunized with T4-bTg had antibody titers from 1:4000 or 1:8000, whereas the rabbits vaccinated with T3-bTg had titers from 1:10000 to 1:40000. Two of the T4-antisera revealed maximal T4-binding between pH 3.8 and 4.2 with affinity constants of 7.8 X 109 1/mol and 6.0 X 109 1/mol. With these two antisera a simple, rapid, protein-independent T4 radioimmunoassay at pH 4.0 in unextracted serum was established without using thyroxine binding globulin blocking agents. Precision of the T4 radioimmunoassay is documented by an interassay coefficient of variation (CV) calculated from 10 different assays of 5.7 % at a mean T4 concentration of 43 Mg/1 T4,3.5 % at 76 jug/1 T4 and 6.4 % at 165 jug/1 T4. The intra-assay CV was calculated to be 3.7%, 2.8 % and 4.7 % at these concentrations. Accuracy of the T4-RIA is documented by identi-cal standard curves when T4 standards were diluted in 1 g/1 human serum albumin in phosphate buffer, in 40 g/1 human serum albumin in phosphate buffer, or in T4-free serum, and by recovery studies and dilution curves of hyperthyroid patients. Three T3 antisera with an optimal binding of T3 at pH 4 and three T3 antisera with a pH optimum in the pH range from 8.5 to 10.0 were obtained. A thyroxine binding globulin independent radioimmunoassay for T3 could b