Use of the monoclonal antibody DAKO-ER beta (8D5-1) to measure oestrogen receptor beta in breast cancer cells

Background: Oestrogen receptor beta (ER beta) is highly homologous with the classical ER (known now as ER alpha). The exact role of ER beta in breast cancer and its contribution in influencing patient response to endocrine th erapy remains unclear. The aim of this study was to develop and evaluate a flow cytometric method for the detection of ER beta in breast cancer cells using the DAKO monoclonal anti-ER beta 8D5-1 antibody. Methods: MCF7 cells were used as a positive control and U937 as a negative control for titration of the antibody. Cell lines and tumour samples were f ixed with 1% paraformaldehyde and permeabilised with 0.5% saponin prior to flow cytometric analysis. Results: A ten fold difference in expression of ER beta within the differen t breast cell lines studied was found. Confirmation of antibody specificity against ER beta protein by Western blot analysis detected a single band at approximately 65kDa. ER beta immunopositive nuclei were identified in MCF7 cells by immunohistochemistry. Conclusions: DAKO ER beta 8D5-1 antibody is specific for ER beta protein an d does not cross react with ER alpha protein. Using this antibody, ER beta can be detected and accurately quantified in cell lines and solid breast tu mours by flow cytometry. Cytometry 45:65-72, 2001. (C) 2001 Wiley-Liss, Inc .