Production of Antibodies Specifically Binding Triiodothyronine and Thyroxine

In an attempt to obtain antisera for radioimmunoassay of thyroid hormones, we immunized rabbits with various thyroid fractions, various conjugates of triiodothyronine (T3), or a charcoal-T3 complex. We examined the frequency of appearance of antibodies which bind T3 and/or thyroxine (T4) and the potency of these antibodies. Human thyroglobulin was the most effective antigen when injected in Freund's adjuvant but a poor one when injected in saline. Human thyroid microsomal fraction was more effective than thyroglobulin in saline, rabbit thyroid microsomal fraction, the various T3 conjugates, or the charcoal-T3 complex. Antisera produced by immunization against human thyroglobulin (HTg) possessed 2 distinct sets of binding sites, one which was more specific for T3 than T4 and one vice versa. In some antisera, the titer of the “T3-specific” site was higher, while in others the reverse was true, although rabbits had been immunized against the identical thyroglobulin. There was also no correlation between the T3-binding activity of a serum and its anti-HTg hemagglutinating titer. The potential application of these antisera in measurement of thyroid hormones by radioimmunoassay has been studied. The standard curve for T3 was linear from 0.1 to 10 ng/assay tube and cross reaction with T4 varied from 0.8 to 2.5%, depending upon the conditions used. Cross reaction with MIT, DIT, or tetraiodothyroacetic acid was negligible. Thyroglobulin did not displace labeled T3 when tested in amounts varying from 0.1 to 100 ng/assay tube. The standard curve for displacement of labeled T4 by unlabeled T4 was linear from 0.5 to 100 ng and cross reaction with T3 in 2 antisera thus tested was approximately 5 and 8%, indicating that concentrations of T3 likely to be present in serum would not interfere appreciably. These studies indicate that immunization against HTg may be a practical method for the production of thyronine-binding antibodies for radioimmunoassay of T4 and T3.