Method development and interspecies comparison of estrogen receptor binding assays for estrogen agonists and antagonists

We have used gel filtration chromatography to separate specifically bound from nonspecifically bound and unbound radioligand in estrogen receptor binding assays. Relative binding affinities (RBAs) were obtained by the competition method, using [H-3]-estradiol as the radioligand. Mixtures of environmental estrogens behaved additively in the assay, and in an interspecies comparison, the order of affinity was similar in rat liver and trout liver. Similar RBAs in different species are supported by sequence alignment analysis, which indicates conservation of key amino acid residues and sequences in the ligand binding domains of estrogen receptors of diverse species. The results are discussed in the context of the requirement for screening assays for endocrine modulators.