Phage display-derived antibody fragments against conserved regions of VacA toxin of Helicobacter pylori

Phage display-derived antibody fragments against conserved regions of VacA toxin of Helicobacter... Infection with Helicobacter pylori may result in the emergence of gastric adenocarcinoma. Among various toxins assisting pathogenesis of H. pylori, the vacuolating cytotoxin A (VacA) is one of the most potent toxins known as the major cause of the peptic ulcer and gastric adenocarcinoma. To isolate single-chain variable fragments (scFvs) against two conserved regions of VacA, we capitalized on the phage display technology and a solution-phase biopanning (SPB). Characterization of scFvs was carried out by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and surface plasmon resonance (SPR). Bioinformatics analyses were also performed in order to characterize the structural and functional properties of the isolated scFvs and the interaction(s) between the isolated antibodies (Ab)-antigen (Ag). After four rounds of biopanning, the positive colonies detected by scFv ELISA were harvested to extract the plasmids and perform sequencing. Of several colonies, three colonies showed high affinity to the VacA1 and two colonies for the VacA2. Further complementary examinations (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot, SPR, and flow cytometry) displayed the high affinity and specificity of the isolated scFvs to the VacA. Docking results revealed the interaction of the complementarity-determining regions (CDRs) with the VacA peptide. In conclusion, for the first time, we report on the isolation of several scFvs against conserved residues of VacA toxin with high affinity and specificity, which may be used as novel diagnostic/therapeutic tool in the H. pylori infection. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Microbiology and Biotechnology Springer Journals

Phage display-derived antibody fragments against conserved regions of VacA toxin of Helicobacter pylori

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Publisher
Springer Journals
Copyright
Copyright © 2018 by Springer-Verlag GmbH Germany, part of Springer Nature
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
ISSN
0175-7598
eISSN
1432-0614
DOI
10.1007/s00253-018-9068-4
Publisher site
See Article on Publisher Site

Abstract

Infection with Helicobacter pylori may result in the emergence of gastric adenocarcinoma. Among various toxins assisting pathogenesis of H. pylori, the vacuolating cytotoxin A (VacA) is one of the most potent toxins known as the major cause of the peptic ulcer and gastric adenocarcinoma. To isolate single-chain variable fragments (scFvs) against two conserved regions of VacA, we capitalized on the phage display technology and a solution-phase biopanning (SPB). Characterization of scFvs was carried out by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and surface plasmon resonance (SPR). Bioinformatics analyses were also performed in order to characterize the structural and functional properties of the isolated scFvs and the interaction(s) between the isolated antibodies (Ab)-antigen (Ag). After four rounds of biopanning, the positive colonies detected by scFv ELISA were harvested to extract the plasmids and perform sequencing. Of several colonies, three colonies showed high affinity to the VacA1 and two colonies for the VacA2. Further complementary examinations (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot, SPR, and flow cytometry) displayed the high affinity and specificity of the isolated scFvs to the VacA. Docking results revealed the interaction of the complementarity-determining regions (CDRs) with the VacA peptide. In conclusion, for the first time, we report on the isolation of several scFvs against conserved residues of VacA toxin with high affinity and specificity, which may be used as novel diagnostic/therapeutic tool in the H. pylori infection.

Journal

Applied Microbiology and Biotechnology – Springer Journals

Published: Jun 3, 2018

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