Characterization of mAb6-9-1 monoclonal antibody against hemagglutinin of avian influenza virus H5N1 and its engineered derivative, single-chain variable fragment antibody

Róża Sawicka , Paweł Siedlecki , Barbara Kalenik , Jan P. Radomski , Violetta Sączyńska , Anna Porębska , Bogusław Szewczyk , Agnieszka Sirko , Anna Góra-Sochacka

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.
Author Róża Sawicka
Róża Sawicka,,
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, Paweł Siedlecki
Paweł Siedlecki,,
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, Barbara Kalenik
Barbara Kalenik,,
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, Jan P. Radomski
Jan P. Radomski,,
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, Violetta Sączyńska
Violetta Sączyńska,,
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, Anna Porębska
Anna Porębska,,
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, Bogusław Szewczyk ZSzR
Bogusław Szewczyk,,
- Laboratory of Recombinant Vaccines
, Agnieszka Sirko
Agnieszka Sirko,,
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, Anna Góra-Sochacka
Anna Góra-Sochacka,,
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Journal seriesActa Biochimica Polonica, ISSN 0001-527X
Issue year2017
Vol64
No1
Pages85-92
Publication size in sheets0.5
Keywords in Englishinfluenza virus, monoclonal antibody, scFv, hemagglutinin, antigenic epitope
DOIDOI:10.18388/abp.2016_1292
URL http://www.actabp.pl/pdf/Preprint/2016_1292.pdf
Languageen angielski
LicenseJournal (articles only); published final; Other open licence; with publication
Score (nominal)15
ScoreMinisterial score = 15.0, 20-12-2017, ArticleFromJournal
Ministerial score (2013-2016) = 15.0, 20-12-2017, ArticleFromJournal
Publication indicators WoS Impact Factor: 2016 = 1.159 (2) - 2016=1.491 (5)
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