Development of ELISA methodologies for the direct determination of 17β-estradiol and 17α-ethinylestradiol in complex aqueous matrices

Abstract

This study comprises the development of enzyme-linked immunosorbent assays (ELISAs) for the quantification of 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) in complex aqueous matrices without any sample clean-up procedures. Salinity and dissolved organic matter were selected as potential interfering agents in the analysis of E2 and EE2. The optimization was performed in order to (i) overcome matrix effects, and to (ii) increase sensitivity. The addition of a sample buffer containing bovine serum albumin (BSA) prior to the sample was found to decrease the influence of matrix effects. Moreover, adjustments of this buffer's pH together with the optimization of tracer (T) dilution and incubation time were undertaken in order to lower the quantification range. The optimized methods allowed the quantification of E2 and EE2 in the ranges 0.03-200μgL-1and 0.02-10μgL-1, respectively. The assays were applied to real aqueous samples. It was possible to do a first approach to the levels of E2 in Portuguese surface and waste waters; however, it was not feasible to detect EE2 in the samples tested.