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Clinical Chemistry 18: 212-216, 1972;
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Clinical Chemistry, Vol 18, 212-216, Copyright © 1972 by the American Association for Clinical Chemistry

Measurement of Unconjugated 17beta-Estradiol in Plasma by Competitive Protein Binding

B. S. Knox 1 and J. T. France 1

1 Postgraduate School of Obstetrics and Gynaecology, The University of Auckland, Auckland 3, New Zealand.

A sensitive and quick competitive protein-binding method is described for measuring unconjugated 17beta-estradiol in the plasma of women and sheep. One-milliliter aliquots of plasma, with tritiated estradiol standard added as an index to recovery, are extracted with methylene chloride. Plasma extracts of 17beta-estradiol are purified and isolated by adsorption on G-15 Sephadex. The purification procedure does not separate 17agr-estradiol from 17beta-estradiol, but there is less than 0.05% cross reaction in the binding assay. Recovery of 17beta-estradiol is 50-55%. Plasma from human twin pregnancies is used as a source of binding protein in the displacement assay. Free 17betaestradiol is separated from bound estradiol by ammonium sulfate precipitation. The standard curve lies in the range 0-200 pg, with less than 4% variation between duplicate points. Sensitivity of the displacement curve at zero value is 2 pg. Values for method blanks are 9.8 ± 1.3 pg/ml (SD) (n = 28). Variation between replicates, including between-assay variation, is less than 10%.


Key Words: Sephadex chromatography • picogram estimates • sheep • variations during menstrual cycle

Accepted on December 16, 1971






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Copyright © 1972 by the American Association for Clinical Chemistry.