Detection of Epstein-Barr virus-encoded BARF1 protein in vivo

Epstein-Barr virus (EBV) BARF1 (BamHI-A rightward frame 1) protein is a viral oncogen and immunomodulator. BARF1 mRNA is highly and selectively expressed in nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC), however, evidence for presence of BARF1 protein in situ is lacking. In vitro, the hexameric form of BARF1 is rapidly secreted (sBARF1). Secretion of BARF1 from NPC tumor cells may result in the presence of sBARF1 in sera of NPC patients. This study describes the development of new monoclonal and polyclonal antibodies that strongly and selectively bind to ‘native’ NPC-derived sBARF1 protein in its natural hexameric conformation. Using these antibodies, a capture enzyme linked immunosorbent assay (ELISA) was developed to detect sBARF1 protein in sera of NPC patients. Although a detection limit of 10 ng/ml sBARF1 in serum was reached for the ELISA, only 3 of 71 NPC patients at diagnosis showed elevated levels of sBARF1 protein. Possibly sBARF1 is complexed with serum proteins shielding antibody recognition sites. However, sample denaturation prior to ELISA testing did not affect the levels observed. A MALDI-MS/MS approach was developed to overcome this problem, yielding a specific BARF1 identifier peptide. However, the sensitivity of the current setup was less than BARF1-capture ELISA. The BARF1 identifier peptide can be used to further develop a quantitative targeted proteomics approach. Using immunohistochemical staining with various anti-BARF1 antibody reagents, BARF1 could be detected in paraffin-embedded BARF1 expressing cell lines revealing a cytoplasmic localisation. Unfortunately, in patient material, no BARF1 protein expression could be demonstrated. In conclusion, the sensitive sBARF1 capture ELISA (10 ng/mL serum) demonstrated only low levels of sBARF1 in NPC patient sera, conflicting with previously published data. A higher sensitivity of both ELISA and proteomic approach is needed for a diagnostic tool. sBARF1 remains a potential serum biomarker for NPC and GC and its diagnostic value using a targeted proteomics approach should be further evaluated. Chapter 8 EBV BARF1 detection