OBJECTIVES: To develop immunofluorometric procedures for measuring BRCA1 protein in various biological fluids and tissue extracts. DESIGN AND METHODS: Five commercially available monoclonal and polyclonal antibodies against BRCA1 were evaluated for developing competitive and non-competitive immunofluorometric procedures for BRCA1. Biotinylated and nonbiotinylated peptides were used to assess the specificity of the antibodies for blocking experiments and for the competitive immunoassay. Extensive studies to exclude cross-reactivity and non-specific effects in the non-competitive immunoassay were undertaken. Seminal plasmas as well as breast tumor extracts, amniotic fluids and cerebrospinal fluids were analyzed. RESULTS: We designed novel methods for measuring BRCA1 immunoreactivity. One configuration based on the "sandwich-type" immunoassay principle was used for further studies. We discovered that seminal plasma contains an immunoreactive protein which appears to possess the D-20 (aminoterminal) and C-20 (carboxyterminal) epitopes of BRCA1. Molecular weight identification using gel filtration chromatography has shown that the immunoreactive species has a molecular weight between 660 and 160 KDa. CONCLUSIONS: We identified for the first time a protein in seminal plasma that shares immunoreactive epitopes with the BRCA1 tumor suppressor protein. We are currently purifying this protein in order to examine if it is homologous or identical to BRCA1.
OBJECTIVES: To develop immunofluorometric procedures for measuring BRCA1 protein in various biological fluids and tissue extracts. DESIGN AND METHODS: Five commercially available monoclonal and polyclonal antibodies against BRCA1 were evaluated for developing competitive and non-competitive immunofluorometric procedures for BRCA1. Biotinylated and nonbiotinylated peptides were used to assess the specificity of the antibodies for blocking experiments and for the competitive immunoassay. Extensive studies to exclude cross-reactivity and non-specific effects in the non-competitive immunoassay were undertaken. Seminal plasmas as well as breast tumor extracts, amniotic fluids and cerebrospinal fluids were analyzed. RESULTS: We designed novel methods for measuring BRCA1 immunoreactivity. One configuration based on the "sandwich-type" immunoassay principle was used for further studies. We discovered that seminal plasma contains an immunoreactive protein which appears to possess the D-20 (aminoterminal) and C-20 (carboxyterminal) epitopes of BRCA1. Molecular weight identification using gel filtration chromatography has shown that the immunoreactive species has a molecular weight between 660 and 160 KDa. CONCLUSIONS: We identified for the first time a protein in seminal plasma that shares immunoreactive epitopes with the BRCA1tumor suppressor protein. We are currently purifying this protein in order to examine if it is homologous or identical to BRCA1.
Authors: K Charalabopoulos; A Kotsalos; A Batistatou; A Charalabopoulos; P Vezyraki; D Peschos; V Kalfakakou; A Evangelou Journal: Br J Cancer Date: 2006-08-01 Impact factor: 7.640