Radioimmunoassay of Unconjugated and Total Serum Estetrol Using a ¹²⁵I-Iodinated Tracer

Abstract

A radioimmunoassay (RIA) for the measurement of both unconjugated and total serum estetrol has been developed, using an antiserum to an E₄-3-conjugate and a ¹²⁵I-radioiodinated E₄ tracer. Assay of dried ethyl ether extracts was used for the determination of unconjugated E₄, while a direct measurement of unextracted hydrolyzed serum in the presence of 0.3% 8-anilino-1-naphthalene sulphonic acid (ANS) proved adequate for total E₄. Assay reliability was evaluated and the procedure standardized through a series of tests aimed at assessing accuracy, sensitivity and precision. No steroidal interference was found to practically affect the assay (0.3% estriol cross-reactivity), nor were solvent and sample blanks observed in the case of unconjugated E₄. For total E₄ assay, the sample blank effects were acceptably overcome by using hydrolyzed male serum and 0.3% ANS, as a standard diluent. An interassay variability amounting to approximately 10 and 6% resulted for unconjugated E₄ and total E₄ RIA, respectively. A number of serum samples (285 for unconjugated E₄, 147 for total E₄) randomly collected throughout normal pregnancy were assayed. The unconjugated E₄ levels at 15 th week and at term were 62.7 ± 22.6 and 766.5 ± 208.2 (SD) pg/ml, respectively. Total E₄ was about 6-7 times higher than the levels of free E₄ and increased 7 times from the 15th week to term.

Estetrol: A unique steroid in human pregnancy

“…As specific radioimmunoassays for E 4 were developed [24][25][26][27][28][29] it became possible to quantitate plasma levels, and a number of laboratories measured the levels of E 4 in maternal and fetal plasma and amniotic fluid. Table 1 summarizes reported results of unconjugated serum E 4 concentrations in the maternal and fetal circulation.…”