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125I-radioimmunoassay for unconjugated estriol in serum of pregnant women.

An 125I-radioimmunoassay is described which measures unconjugated estriol in the serum of pregnant women. Estriol is extracted into ethyl acetate/hexane, an aliquot is evaporated, and the residue is redissolved in phosphate buffer. The sample is incubated with tracer and antibody at 37 degrees C for 15 min and then at 4 degrees C for 1 h and 45 min. The antibody-bound fraction is then precipitated with polyethylene glycol and isolated by centrifugation. Because the antigen-antibody complex is stable in the presence of polyethylene glycol, the separation steps are not influenced by timing. Extraction recovery of 3H-labeled estriol added to a pool of sera from pregnant women averaged 96.7% (SD = 1.3, n = 10). Estriol-supplemented (5, 10, and 20 microgram/liter) serum from men, carried through the entire procedure, showed analytical recovery ranging from 94 to 106%. Structurally analogous steroids normally present in serum of pregnant women exhibit negligible cross reactivity. Day-to-day precision (CV) is 13.3% (3.1 microgram/liter), 6.4% (7.6 microgram/liter), and 5.6% (21.4 microgram/liter) for n = 21. The current reagent cost (about 17 cents per tube), and a total procedural time, including counting, of 5.5 h make this an acceptable assay for routine use.