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Characterization and use of monoclonal antibodies against bilitranslocase and its determination in clear cell renal cell carcinoma

Abstract

With its worldwide incidence of about 300 000 new cases per year, clear cell Renal Cell Carcinoma (ccRCC) is the seventh most commonly diagnosed cancer in men and the ninth most commonly diagnosed cancer in women. At the hereditary and molecular levels recent research efforts describe large molecular profiling analyses for the molecular cause to be related with the development of ccRCC. Historically, von Hippel-Lindau (VHL) tumor suppressor protein located on chromosome 3p25, the loss of activity of which leads to a syndrome connected with diseases including ccRCC, was among the top genetic causes known. Monoclonal antibodies are an important tool in diagnostics and research, especially when we are dealing with a protein marker of unknown primary structure as in case of bilitranslocase (BTL). BTL is expressed on kidney cells, where it acts as an organic anion transporter. We have shown that there are differences in bilitranslocase expression in normal kidney cells versus early grade kidney cancer. A set of hybridoma cell lines producing antipeptide monoclonal antibodies against segments 235-246 (peptide B) and 298-310 (peptide C) of predicted primary structure of bilitranslocase was cloned by limiting dilution. With a sequence of immune tests we characterized monoclonal antibodies, and used them as a tool to distinguish between grades in progression of ccRCC. We developed monoclonal antibodies against extra- (peptide B) and intra-cellular (peptide C) domains of bilitranslocase protein model. Our results are showing that these antibodies can be used in different immunoassays. Furthermore specificity and affinity of our mAbs allowed us to assess progressive grades of clear cell renal cell carcinoma and thus introduce a potentially novel tool for the diagnostics of ccRCC

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