Radioimmunoassay of unconjugated and total serum estetrol using a 125I-iodinated tracer

A radioimmunossay (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 15th 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.