title: Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody.
abstract: Hemagglutinin (HA), as a major surface antigen of influenza virus, is widely used as a target for production of neutralizing antibodies. Monoclonal antibody, mAb6-9-1, directed against HA of highly pathogenic avian influenza virus A/swan/Poland/305-135V08/2006(H5N1) was purified from mouse hybridoma cells culture and characterized. The antigenic specificity of mAb6-9-1 was verified by testing its cross-reactivity with several variants of HA. The mimotopes recognized by mAb6-9-1 were selected from two types of phage display peptide libraries. The comparative structural model of the HA variant used for antibody generation was developed to further facilitate epitope mapping. Based on the sequences of the affinity- selected polypeptides and the structural model of HA the epitope was located to the region near the receptor binding site (RBS). Such localization of the epitope recognized by mAb6-9-1 is in concordance with its moderate hemagglutination inhibiting activity and its antigenic specificity. Additionally, total RNA isolated from the hybridoma cell line secreting mAb6-9-1 was used for obtaining two variants of cDNA encoding recombinant single-chain variable fragment (scFv) antibody. To ensure high production level and solubility in bacterial expression system, the scFv fragments were produced as chimeric proteins in fusion with thioredoxin or displayed on a phage surface after cloning into the phagemid vector. Specificity and affinity of the recombinant soluble and phage-bound scFv were assayed by suitable variants of ELISA test. The observed differences in specificity were discussed.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Róża
authors.initials: R
authors.lastname: Sawicka
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Paweł
authors.initials: P
authors.lastname: Siedlecki
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Kalenik
authors.affiliation: Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University, Warsaw, Poland.
authors.forename: Jan P
authors.initials: JP
authors.lastname: Radomski
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Violetta
authors.initials: V
authors.lastname: Sączyńska
authors.affiliation: Institute of Biotechnology and Antibiotics, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Porębska
authors.affiliation: Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdańsk, Poland.
authors.forename: Bogusław
authors.initials: B
authors.lastname: Szewczyk
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Sirko
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Góra-Sochacka
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D018076:DNA, Complementary; D000939:Epitopes; D006388:Hemagglutinins / Q000276:immunology*; D006825:Hybridomas; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology*; D051379:Mice; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D006388:Hemagglutinins; D019151:Peptide Library; D057127:Single-Chain Antibodies
keywords: antigenic epitope; hemagglutinin; influenza virus; monoclonal antibody; scFv