Development of competitive ELISAs for 17beta-estradiol and 17beta-estradiol +estrone+estriol using rabbit polyclonal antibodies.

Estrogens are a family of feminizing hormones that are excreted by vertebrates. It has been documented that their presence in surface waters, even in the ng/L range, can have detrimental impacts on fish reproduction. Two competitive enzyme-linked immunosorbent assays using rabbit polyclonal antibodies were developed: one for 17beta-estradiol and a second one for 17beta-estradiol (E2)+estrone (E1)+estriol (E3). Two different conjugates were synthesized using the Mixed-anhydride (for the 17beta-estradiol ELISA) and the Mannich (for the E1 + E2 + E3 ELISA) reactions. The 17beta-estradiol ELISA was highly specific with an IC(50) of 243 ng/mL for 17beta-estradiol. The E1 + E2 + E3 ELISA exhibited cross-reactivity with estrone (85%) and estriol (62%) with an IC(50) of 18 ng/mL for 17beta-estradiol. Cross-reactivity was tested against 13 chemically related compounds and both immunoassays showed significant cross-reactivity with two estradiol conjugates: beta estradiol-17-valerate and beta estradiol-3-benzoate (from 57 to 84 %) for which, to our knowledge, there are currently no commercially available ELISA. Characteristics (sensitivity, inter and intra assay variation, and cross-reactivity) of the E1 + E2 + E3 ELISA were further compared to those from a commercial Estriol ELISA. The commercial ELISA was more specific, sensitive and its inter-assay variation was less (9.5% compared to 10% for the E1 + E2 + E3 ELISA) but the E1 + E2 + E3 ELISA had less intra-assay variation (4% compared to 5% for the commercial ELISA). Finally, a solid-phase extraction method compatible with the E1 + E2 + E3 immunoassay demonstrated that this combined approach of extraction and immunoassay had good potential for determining estrogen concentrations in environmental samples such as surface water in urban and agricultural ecosystems.