Enzyme immunoassay for total oestrogens in pregnancy plasma or serum

We have developed an enzyme immunoassay for total oestrogens in pregnancy serum of plasma, using horseradish peroxidase (HRP) as the marker enzyme. A test combination consisting of an antiserum against oestriol-16/17-monosuccinyl-albumin and oestriol-16/17-monosuccinyl-HRP yielded a sensitive system, which reacted to approximately the same extent with oestrone, oestradiol, oestriol and their 16- and 17-conjugates. Samples had to be diluted 1 to 10 to avoid interference of plasma factors with the immune reaction. Bound/free separation was achieved with the double antibody solid phase (DASP) method. The HRP activity of the bound fraction was measured, after washing, to eliminate plasma factors disturbing the HRP reaction. The detection limit of the assay system was approx. 0.1 pmol/tube, while the index of precision λ ranged from 0.02 to 0.06. To measure total oestrogens, including the 3-conjugated ones, we used an enzymatic hydrolysis with an extract of Helix pomatia. Hydrolysis was found to be optimal after 1 h at 50°C and pH 5.0. The method was used on serum samples from normal pregnancies. The results showed a very good correlation ( r = 0.98) with those obtained by radioimmunoassay. Normal values for total oestrogens during pregnancy were determined in a multicentre clinical trial.