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A methodological approach to development of a radioimmunological system for quantitative assay of thyroxine (T4) in human blood serum is described. It implies investigation of every component of the system and matching of the components by the physico-chemical properties and concentrations. An antiserum to the T4 conjugate was prepared with bovine serum albumin. The hapten "fitting density" was 30 mol per 1 mol of protein. The antiserum had a titer of 5000, was highly specific and showed affinity to T4, Ka (7.6 +/- 1.0) X 10(8) M-1. The characteristics of the antiserum did not depend on concentration of the hydrogen ions in solution at pH 6.0-9.0 and the ionic strength of the buffer solutions within M 0.05-1.0. 8-Aniline-1-naphthalene sulfonic acid (ANA) in concentrations of 0.05-0.1 per cent completely inhibited binding of T4 to endogenic proteins of the blood serum and had no effect on the antiserum interaction with T4. This provided performance of the radioimmunological assays at 0.1 per cent concentration of ANA. [125I]T4 with the specific activity of 40 TBk/g was used in the study. The quality of [125I] T4 was estimated by its immune reactivity. The standard solutions of T4 were prepared with donor blood serum as the basis. It was shown that quantitative assays of T4 in blood serum within clinically significant ranges of the hormone concentration are possible at 1:5000 final dilution of the antiserum, the [125I] T4 activity of 20 000-30 000 imp/min per a specimen and 5 per cent content of the blood serum in the analytical specimen. A simple and reliable technique for T4 assay in human blood serum is developed.