1. Detection sensitivity
0,00
0,20
0,40
0,60
0,80
1,00
1,20
1,40
1,60
#20+#42 #20+#42+BT
Absorbance
sBARF1 300ng/ml
sBARF1 100ng/ml
sBARF1 50ng/ml
sBARF1 25ng/ml
sBARF1 10ng/ml
pool human serum
Secreted BARF1 protein in sera of NPC patients
293HEK/Rat #42 293HEK BARF1/Rat #42
Hedyani Juwana, Eveline Hoebe, George Scheffer, Karin van Schie, Erik Hopmans,
Astrid Greijer, Jaap Middeldorp
Department of Pathology, VU University Medical Center, The Netherlands
Results
BARF1 protein levels in sera of NPC patients were compared to sera of
healthy EBV positive and EBV negative donors. 10% of NPC sera
showed sBARF1 concentrations above the cut-off value. sBARF1
concentrations in NPC sera were not significantly different compared to
controls.
Conclusion
New MoAbs to hexamer sBARF1 were made. MoAbs can detect
BARF1 by IHC and immunoblot. Sensitivity of assay to detect sBARF1
is improved up to 10 ng/ml. Most NPC sera have sBARF1 concentration
lower than the cut-off value. We conclude that sBARF1 proteins is not
present at significant levels in blood nor detectable in NPC FFPE
tissue, even though BARF1 RNA is highly expressed in NPC.
Background research
The Epstein-Barr virus BamHI-A rightward frame1 (BARF1) is a
EBV oncogene, which is highly transcribed in undifferentiated
Nasopharyngeal Carcinoma (NPC).
In vitro, the hexameric form of BARF1 (±180 kDa) is rapidly
secreted as sBARF1 in culture medium. In vivo BARF1 protein
might also be secreted in serum from NPC tumor, since antibodies
against BARF1 were detected in sera of NPC patients. Previously
BARF1 protein was also found in sera of NPC patients in high
amount (Houali et al., 2007), but remained unconfirmed, despite
using identical monoclocal antibodies (MoAbs) to monomeric
BARF1, i.c. mouse 4A6 and 6F4.
AIM
1. Develop new rat MoAbs that can recognize the NPC-derived
sBARF1 protein in its natural hexameric formation.
2. Optimize the detection and quantification of sBARF1 in sera of
NPC patients using antigen-capture sandwich ELISA.
Monoclonal antibodies against BARF1
New MoAbs were produced by immunizing rats with purified NPC-
derived hexameric sBARF1 protein. Hybridomas were analyzed by
immunoblot and sandwich ELISA. Hybridomas producing MoAbs to
hexameric sBARF1 in immunoblot and recognized sBARF1 in
ELISA were selected. Hybridomas were also tested in IHC besides
4A6 and 6F4. IHC on BARF1-HEK was positive, but BARF1
staining in NPC tissues remained negative as before with 6A4 and
4F6 MoAbs.
293HEK BARf1/Rat #20293HEK/Rat #20
Cut-off value (28 ng)
Detection sBARF1 in NPC sera
Sandwich ELISA using Rat #20 as capture antibody and Rat #42 as
detection antibody was selected as the best method to detect BARF1
protein present in serum. After enhancement of the detection signal with
a tyramide amplification, a sensitivity of 10 ng BARF1/ml serum was
reached in human pool serum background.