[Screening of dominant epitopes of antigens related to antibodies in the sera of patients with nasopharyngeal carcinoma].
OBJECTIVE
Epstein-Barr virus (EBV) is closely related to nasopharyngeal carcinoma (NPC). Many kinds of methods can be used to examine antibodies in NPC patients sera. This study was to screen the dominant epitopes from random peptide libraries (RPLs) displayed on phage using the EBV-related antibodies purified from the sera of NPC patients, and find new antigens at the epitope level.


METHODS
The EBV-related antibodies were eluted from the sera of NPC patients using B95-8 cell EBV proteins as antigen, and the phages from 12-mer RPLs were elutriated for 3 rounds with the antibodies. The positive clones were gained by sandwich ELISA, and competitive inhibition assay from the third elution. The positive clones were sequenced, and the peptide coded by the inserted DNA were blasted with the antigen region of EBV proteins.


RESULTS
Sixty-four phage clones were randomly picked up from the third eluate,25 positive clones were picked up using sandwich ELISA assay, the positive percentage was 39.06%. Thirteen clones were picked up for competitive ELISA assay,11 clones showed inhibitory phenomena,the inhibitory rates were between 18.09% and 65.94%. Five positive clones with high absorbency value, and high inhibitory rates were selected out, the sequences of peptides displayed on these clones were -A-T-S-H-L-H-V-R-L-P-W-T- (d15, and d18), -G-S-T-H-K-H-N-H-F-N-K-T- (d19), -K-P-I-H-E-H-P-H-R-F-K-S- (e8), -H-T-H-K-I-K-I-P-L-P-I-Q- (e23). These peptide sequences showed similarity with the amino acid sequences located in antigen regions of EBV integral membrane protein (d15, and d18), EBV thymidine kinase (d19), and EBV major capsid protein (e8, and e23).


CONCLUSIONS
EBV-related epitopes could be obtained by screening the phages from RPLs with polyclonal antibodies purified from the sera of NPC patients, which may offer new methods of antigen preparation for sera diagnosis of NPC.
Zhang
Ying
Y
Experimental Research Department, Cancer Center, Sun Yat-sen University, Guangzhou, Guangdong, 510060, PR China. zhy99_0@163.com
Xiao
Xi-Bin
XB
Zhang
Chang-Qing
CQ
Li
Jing-Lue
JL
Sun
Yun
Y
Ye
Yong-Zhao
YZ
Feng
Kai-Tao
KT
D000595:Amino Acid Sequence; D000912:Antibodies, Neoplasm / Q000032:analysis* / Q000235:genetics; D000914:Antibodies, Viral / Q000032:analysis* / Q000235:genetics; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology; D036022:Capsid Proteins / Q000235:genetics; D000939:Epitopes / Q000032:analysis* / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009303:Nasopharyngeal Neoplasms / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D017386:Sequence Homology, Amino Acid; D013937:Thymidine Kinase / Q000235:genetics
D000912:Antibodies, Neoplasm; D000914:Antibodies, Viral; D000951:Antigens, Neoplasm; D000956:Antigens, Viral; D036022:Capsid Proteins; D000939:Epitopes; D008565:Membrane Proteins; D019151:Peptide Library; D013937:Thymidine Kinase