Preparation and characterization of monoclonal antibodies against mouse estrogen-enhanced transcript
Objective In order to explore the tissue and sub-cellular distribution of mouse estrogen-enhanced transcript(mEET) protein, we prepared and characterized monoclonal antibodies against mEET. Methods Hybridomas were generated by fusion of Sp2/0 myelomas and spleen cells from rats immunized with mEET recombinant proteins. Hypoxanthine-aminopterin-thymidine-resistant clones were then screened for secretion of antibodies specific for mEET by ELISA and identificated by Western blot. The monoclonal antibodies were subsequently used for studies of tissue distribution and sub-cellular localization of mEET protein by immunofluorescence or immunohistochemistry. Results Four hybridoma cell lines, DA10, DB4, 4A5 and 4D8, were produced, each of which stably secrets antibodies again mEET. Isotyping indicated that they were IgM antibodies. Immunofluorescent assay demonstrated that mEET protein was mainly distributed in cytoplasm, whereas immunochemistry study showed wide tissue distribution with particularly high levels of expression in megakaryocytes in bone marrow. Conclusion The monoclonal antibodies against mEET protein have been successfully prepared, which should provide useful reagent for further investigation into the biological function of mEET.