Triiodothyronine Radioimmunoassay

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Abstract
Highly specific antisera to triiodothyronine (T3) were prepared by immunization of rabbits with T3-bovine serum albumin conjugates. Antisera with T3 binding capacity of up to 600 ng/ml were obtained. The ability of various thyronine derivatives to inhibit the binding of T3-125I to anti-T3 serum was found to vary considerably. l-T3, d-T3 and several triiodoanalogues were potent inhibitors of the reaction. Little inhibition of T3-125I binding was produced by l-thyroxine (T4) or other tetraiodo- analogues, thyronine or iodotyrosines. Chromatography of several T4 preparations indicated that most of their very slight activity could be ascribed to contamination with T3. Successful assay of T3 in serum was accomplished by the addition of diphenylhydantoin to the assay system. Under these circumstances, recovery of T3 added to serum was excellent, and addition of T4 was without significant effect. Serum T3 concentrations in normal subjects averaged 145 ±25 ng/100 ml (sd). Increased concentrations (429 ±146 ng/100 ml) were observed in hyperthyroid patients whereas those with hypothyroidism had serum T3 levels of 99 ±24 ng/100 ml. Elevated T3 concentrations were found also in hypothyroid patients receiving 25 μg or more of T3 daily and in those receiving 300 μg of T4 daily. Serial measurements of T3 concentrations in subjects after oral T3 administration revealed peak T3 concentrations 2-4 hr after T3 administration. Intramuscular administration of thyrotropin (TSH) resulted in earlier and more pronounced increases in serum T3 than in serum T4 concentrations. Triiodothyronine (T3)1 was recognized to be a biologically active secretory product of the thyroid gland over a decade ago (1). Recent studies have indicated that it is formed extrathyroidally as well (2, 3). Nevertheless, relatively little information concerning the role of T3 secretion in different thyroid disorders has been accumulated until very recently. Methods for the measurement of T3 which require its extraction from plasma, and often its separation from thyroxine as well, have been described by several investigators (4-11). These methods have proven useful, but they are relatively complicated, the number of samples that can be assayed is limited, and they may be affected by in vitro deiodination of thyroxine. More recently the radioimmunoassay technique has been applied to the measurement of T3. Several preliminary reports have appeared describing the preparation of antibody to triiodothyronine by immunization of animals with T3-protein conjugates and its use for the measurement of T3 in serum (12-15). The present report describes the development of a radioimmunoassay for the measurement of T3, studies of the specificity of the anti-T3 serum, and some initial studies which indicate that the method is applicable to the measurement of T3 in unextracted serum.