Production and Evaluation of High-Quality Thyroxine Antisera for Use in Radioimmunoassay

Abstract

High-quality thyroxine (T4) antisera were raised in rabbits. Conjugates of protein and T4 or T4 methyl ester were given intracutaneously in Freund's complete adjuvant at one or two sites on the back at 1/2- to 3-month intervals. Eleven of 16 immunized rabbits produced antiserum with titers higher than 2 commercial antisera and 4 antisera from other laboratories. All 16 antisera had equilibrium constants (K) higher than the 6 reference sera. The highest titer observed was 130,000 with 88 fmol labeled T4 per ml incubate. A drastic increase in K from about 4 × 108 to 5.8 × 1010 I/mol was observed as the immunization proceeded. Cross-reactivity with triiodothyronine (T3) varied but was generally low. Suppression of the T4 synthesis was attempted by giving 30 nmol (20 μg) T3 per os once a day. Antiserum from rabbits given T3 suppression contained less iodine than antiserum from nonsuppressed control rabbits. The possible reasons for the successful production of the high-quality antisera are discussed.

Key Words:

• Conjugates
• cross-reaction
• dissociation rate constant
• equilibrium constant
• immunization technique
• iodine
• suppression
• titer
• triiodothyronine