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


CONCLUSIONS
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.
Scholler
Nathalie
N
Center for Research on Early Detection and Cure of Ovarian Cancer, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. naths@mail.med.upenn.edu
Gross
Jennifer A
JA
Garvik
Barbara
B
Wells
Lance
L
Liu
Yan
Y
Loch
Christian M
CM
Ramirez
Arturo B
AB
McIntosh
Martin W
MW
Lampe
Paul D
PD
Urban
Nicole
N
D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D014408:Biomarkers, Tumor / Q000097:blood*; D019921:Biotinylation; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins / Q000097:blood; D006801:Humans; D009367:Neoplasm Staging; D010051:Ovarian Neoplasms / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology; D049488:Phosphatidylethanolamine Binding Protein / Q000097:blood / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility; D015003:Yeasts; D014841:von Willebrand Factor / Q000276:immunology
D000906:Antibodies; D014408:Biomarkers, Tumor; C426245:EMILIN2 protein, human; D006023:Glycoproteins; C511482:PEBP1 protein, human; D049488:Phosphatidylethanolamine Binding Protein; D011994:Recombinant Proteins; D014841:von Willebrand Factor