Determination of the fine epitope specificity of an anti-hepatitis B virus X protein monoclonal antibody using microanalytical and molecular biological methods.
The recombinant form of the 17kDa, highly hydrophobic and disulfide-bonded hepatitis B virus X protein (HBX) was used for developing a set of monoclonal antibodies (Mab). Our present goal was to determine the fine epitope specificity of our anti-HBX Mab. Based on computer analysis two sequences (amino acids 22-31 and 100-114) were predicted for possessing high immunogenity while the anti-HBX Mab did not recognized them. Limited proteolysis and mass spectroscopic analysis suggested another possible sequence (amino acids 14-26), which also proved to be negative using an immunoserological test. Subsequently, we performed a screen of a phage displayed random peptide library, by which we could localize the epitope to amino acids 88-93. This finding was confirmed using three overlapping fusion peptides spanning amino acids 77-142. Their testing in ELISA assigned the epitope to amino acids 77-95, which supports the result obtained by screening the phage displayed library. Our results suggest the necessity of a complex application of current molecular biological and immunological techniques in fine structure mapping. This approach will be useful to study the prognostic relevance of different antigenic sites on HBX during the development of chronic hepatitis and primary hepatocellular carcinoma.
Pál
József
J
Department of Immunology and Biotechnology, Faculty of Medicine, University of Pécs, Szigeti u. 12, Pécs H-7643, Hungary.
Czömpöly
Tamás
T
Nyárády
Zoltán
Z
Marczinovits
Ilona
I
Janáky
Tamás
T
Kele
Zoltán
Z
Felici
Franco
F
Németh
Péter
P
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000276:immunology*; D006515:Hepatitis B virus / Q000276:immunology*; D006801:Humans; D051379:Mice; D015534:Trans-Activators / Q000276:immunology*; D054334:Viral Regulatory and Accessory Proteins
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein