Oestradiol-BSA conjugates for receptor histochemistry: problems of stability and interactions with cytosol

The validity of histochemical methods for the localization of hormone receptors based on the binding of fluorescent bovine serum albumin conjugates of oestradiol was examined with respect to their stability and their interactions with the oestrogen receptor type I. Stability was assessed by measuring free oestrogen in conjugates by radioimmunoassay and/or receptor protein binding assay. Sufficient free oestrogen--in order to saturate type I and type II binding sites (ER I, ER II)--was detected in freshly prepared conjugates. This free oestrogen originates in inadequate removal of adsorptively bound original ligand after synthesis. Apart from this fact, conjugates appeared to be unstable in aqueous solutions, especially under the conditions used for for histochemical methods. Free oestrogen extracted from the conjugates was subjected to high performance liquid chromatography. Amongst the eluted peaks, oestradiol and/or the original ligand used for synthesis were identified. The in vitro interaction of conjugates with oestrogen receptors was studied by competitive binding analysis and by incubation of cytosol with a Sepharose-bound conjugate. The results, especially those concerning the amount of free oestrogen, suggest that neither ER I nor ER II is involved in the staining mechanism of conjugates.