Determination of the fine epitope specificity of an anti-hepatitis B virus X protein monoclonal antibody using microanalytical and molecular biological methods

J. Pál, T. Czömpöly, Zoltán Nyárády, Ilona Marczinovits, T. Janáky, Z. Kele, Franco Felici, P. Németh

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)241-246
Number of pages6
JournalMolecular Immunology
Volume40
Issue number5
DOIs
Publication statusPublished - Sep 2003

Fingerprint

Epitopes
Monoclonal Antibodies
Amino Acids
Bacteriophages
Immunologic Techniques
Peptide Library
Protein Sequence Analysis
Chronic Hepatitis
Disulfides
Proteolysis
Libraries
Amino Acid Sequence
Hepatocellular Carcinoma
Enzyme-Linked Immunosorbent Assay
Peptides
hepatitis B virus X protein

Keywords

  • Epitope mapping
  • Hepatitis B virus X protein
  • Mass spectrometry
  • Monoclonal antibody
  • Phage display

ASJC Scopus subject areas

  • Molecular Biology
  • Immunology

Cite this

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abstract = "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.",
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AU - Pál, J.

AU - Czömpöly, T.

AU - Nyárády, Zoltán

AU - Marczinovits, Ilona

AU - Janáky, T.

AU - Kele, Z.

AU - Felici, Franco

AU - Németh, P.

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