A novel enzyme-linked immunosorbent assay for ethynylestradiol using a long-chain biotinylated EE2 derivative

Ethynylestradiol (EE2) is one of the most potent endocrine disrupting compounds capable to induce estrogenic effects even at trace level concentrations in the aquatic environment. Methods for detecting EE2 in such concentrations are generally based on GC or HPLC coupled to at least one mass spectrometer. Another approach are immunoassays and sensor systems but for most designs, derivatives of EE2 are required (e.g. for coupling to carrier proteins, enzyme or fluorescent labels, etc.). Here we present the straightforward synthesis and complete characterization of a new long-chain biotinylated EE2 derivative. The new EE2 derivative is used as tracer in a direct competitive enzyme-linked immunosorbent assay (ELISA) for the determination of EE2. With pure water, the limit of detection (LOD, signal-to-noise ratio, S/N = 3) and the test midpoint were found to be 14 and 136 ng l(-1), respectively. Cross reactivity (CR) was tested for 10 endogenous steroids and the BSA-conjugate used for immunization, as well as a synthetic precursor of the conjugate. Among the naturally occurring compounds, CR was determined to be maximum for metabolites of EE2 conjugated at ring-position 3 (17% and 37% for 3-glucuronide and 3-sulphate, respectively). Assay stability was tested against humic substances and organic solvents. Increasing amounts of organic solvents in the sample caused a clear decrease in sensitivity, presence of humic substances lead to an overestimation of EE2.