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.
Autor Róża Sawicka
Róża Sawicka
-
, Paweł Siedlecki
Paweł Siedlecki
-
, Barbara Kalenik
Barbara Kalenik
-
, Jan P. Radomski
Jan P. Radomski
-
, Violetta Sączyńska
Violetta Sączyńska
-
, Anna Porębska
Anna Porębska
-
, Bogusław Szewczyk (MWB UG i GUMed / IB UG / ZSzR)
Bogusław Szewczyk
- Zakład Szczepionek Rekombinowanych
, Agnieszka Sirko
Agnieszka Sirko
-
, Anna Góra-Sochacka
Anna Góra-Sochacka
-
Tytuł czasopisma/seriiActa Biochimica Polonica, ISSN 0001-527X, (A 15 pkt)
Rok wydania2017
Tom64
Nr1
Paginacja85-92
Objętość publikacji w arkuszach wydawniczych0.5
Słowa kluczowe w języku angielskiminfluenza virus, monoclonal antibody, scFv, hemagglutinin, antigenic epitope
Klasyfikacja ASJC1300 General Biochemistry, Genetics and Molecular Biology
DOIDOI:10.18388/abp.2016_1292
URL http://www.actabp.pl/pdf/Preprint/2016_1292.pdf
Języken angielski
LicencjaCzasopismo (tylko dla artykułów); licence.documentVersion.FINAL_PUBLISHED; Inna otwarta licencja; w dniu opublikowania
Punktacja (całkowita)15
PunktacjaPunktacja MNiSW = 15.0, 24-07-2019, ArticleFromJournal
Wskaźniki publikacji Cytowania WoS = 1; Scopus SNIP (Source Normalised Impact per Paper): 2016 = 0.563; Impact Factor WoS: 2017 = 1.239 (2) - 2017=1.541 (5)
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