A novel nuclear antigen
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
The human oestrogen receptor (hER) mediates some effects of the steroid hormone oestrogen and functions as a ligand-dependent transcription factor in the nuclei of oestrogen-sensitive cells. The measurement of hER levels in breast cancer biopsies provides useful clinical information regarding therapy and prognosis. The current study describes a monoclonal antibody raised against hER aa 497-507 which recognises a novel nuclear antigen. Monoclonal antibodies were raised by immunising mice with a synthetic fragment of the hER (aa 497-507) conjugated to keyhole lymphocyte haemocyanin. Thirty antibody secreting hybridomas were identified. Hybridoma supernatants were characterised by enzyme-linked immunosorbent assay (ELISA), immunological staining using MCF-7 cells, binding studies, and SDS-PAGE Western blotting. One supernatant (15F6) displayed nuclear staining in fixed MCF -7 cells. Staining could be abolished by pre-incubation of the supernatant with the aa 497-507 peptide and peptide conjugates, but not with an unrelated hER peptide (aa 256-275). This antibody also stained the nuclei of hER negative breast cell lines MDA-MB-231 and MDA-MB-330, the breast cell line T47D, and liver cell line HepG2. Immunological staining of human tissue sections reveal the antigen to be present in the nuclei of keratocytes in skin and tubule and luminal endothelial cells of the kidney. The antibody identified a 120 kD band on Western blots with cytosols prepared from human breast cell lines and in solubilised cells. The antibody does not precipitate 16a-iodooestradiol-labelled ER from MCF-7 cells. Expression-linked screening of the MCF-7 cDNA library with antibody 15F6 identified nine positive clones. Antibody staining could be blocked by pre-incubating the antibody with hER aa 497-507-BSA conjugate, but not with an unrelated hER peptide conjugate. The (260 bp) clones were found to be identical. Submission of sequence to BLAST protein and nucleotide databases revealed a lack of homology to known proteins and genes. Sequence was matched to expressed sequence tags (ESTs) from brain, liver/spleen, uterus, ovary, colon, heart, and placenta. To further define the epitope of antibody 15F6, the sequence was translated and three peptides containing potential epitopes, comparable to the hER aa 497-507 region, were synthesised and tested by ELISA. The putative epitope was shown to be contained within one of these peptides.