This assay was developed for use in clinical trials and pharmacokinetic studies of contraceptive and hormone replacement therapy (HRT) preparations. The method is an extraction RIA and not subject to the well-documented interference problems associated with direct serum/plasma immunoassays.

Ethinyl Estradiol in the sample (150µL of serum or plasma) is extracted with diethyl ether. The extract is dried and redissolved in assay buffer. The redissolved extract is assayed for Ethinyl Estradiol by 3H-RIA ( tritium labelled radioimmunoassay with charcoal separation). All samples, standards and quality control specimens are run in duplicate.

The assay is controlled through the use of at least 3 Internal Quality Control samples in every run. In addition the performance of every run is closely monitored for various parameters including imprecision, antibody binding of tracer, non-specific binding and reproducibility of the standard curve.

• Standard Curve: The range of the standard curve is 0 to 33,738 pmol/L (10,000 pg/mL).
• Precision: The between-run precision of the assay is generally less than 10% CV.
• Sensitivity: The assay reliably measures concentrations as low as 337 pmol/L (100 pg/mL).

The cross-reactions of the anti-Ethinyl Estradiol antibody used are shown below:

Substance % Cross Reaction Ethinyl Estradiol 100 Norethisterone (norethindrone) 10.0 17b-Estradiol 0.3 Estrone 0.02 17a-Estradiol 0.008 Diethylstilbestrol 0.004 Hexoestrol 0.004 Dienoestrol 0.004 Zeranol <0.004 17b-Nortestosterone <0.004 17b-Trenbolone <0.004 Estriol <0.004 Methyltestosterone <0.004 Epitestosterone <0.004 Progesterone <0.004 Cortisol <0.004 Testosterone <0.004 Dehydroepiandrosterone <0.004 17a-Hydroxyprogesterone <0.004

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