Use of cancer-specific yeast-secreted in vivo biotinylated recombinant antibodies for serum biomarker discovery

J Transl Med. 2008 Jul 24:6:41. doi: 10.1186/1479-5876-6-41.

Abstract

Background: Strategies to discover circulating protein markers of ovarian cancer are urgently needed. We developed a novel technology that permits us to isolate recombinant antibodies directed against the potential serum biomarkers, to facilitate the further development of affinity reagents necessary to construct diagnostic tests.

Methods: This study presents a novel discovery approach based on serum immunoprecipitation with cancer-specific in vivo biotinylated recombinant antibodies (biobodies) derived from differentially selected yeast-display scFv, and analysis of the eluted serum proteins by electrophoresis and/or mass spectrometry.

Results: Using this strategy we identified catabolic fragments of complement factors, EMILIN2, Von Willebrand factor and phosphatidylethanolamine-binding protein 1 (PEBP1 or RKIP) in patient sera. To our knowledge, this is the first report of a soluble form of PEBP1 in human. Independent evidence for ovarian cancer-specific expression of PEBP1 in patient sera was found by ELISA assays and antibody arrays with anti-PEBP1 antibodies. PEBP1 was detected in 29 out of 30 ascites samples and discriminated ovarian cancer sera from controls (p = 0.02). Finally, we confirmed by western blots the presence of a 21-23 kDa fragment corresponding to the expected size of PEBP1 but we also showed additional bands of 38 kDa and 50-52 kDa in various tissues and cell lines.

Conclusion: We conclude that the novel strategy described here allows the identification of candidate biomarkers that can be variants of normally expressed proteins or that display cancer-specific post-translational modifications.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antibodies / genetics
  • Antibodies / immunology*
  • Biomarkers, Tumor / blood*
  • Biotinylation
  • Case-Control Studies
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Fluorescent Antibody Technique, Indirect
  • Glycoproteins / blood
  • Humans
  • Neoplasm Staging
  • Ovarian Neoplasms / diagnosis*
  • Ovarian Neoplasms / genetics*
  • Ovarian Neoplasms / pathology
  • Phosphatidylethanolamine Binding Protein / blood
  • Phosphatidylethanolamine Binding Protein / genetics
  • Recombinant Proteins / immunology
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Solubility
  • Yeasts*
  • von Willebrand Factor / immunology

Substances

  • Antibodies
  • Biomarkers, Tumor
  • EMILIN2 protein, human
  • Glycoproteins
  • PEBP1 protein, human
  • Phosphatidylethanolamine Binding Protein
  • Recombinant Proteins
  • von Willebrand Factor