BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Phage display-derived antibody fragments against conserved regions
of VacA toxin of Helicobacter pylori
Mohammad Reza Tohidkia
Received: 16 January 2018 /Revised: 1 May 2018 /Accepted: 2 May 2018
Springer-Verlag GmbH Germany, part of Springer Nature 2018
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.
Phage antibody display
Helicobacter pylori is a gram-negative bacterium, which is
colonized in the human stomach. It has infected almost half
of the world’s population, with the highest prevalence in the
developing countries due to poor health and economic back-
grounds (Adler et al. 2014; Lim et al. 2013; Oleastro and
Menard 2013). In patients with H. pylori, in the absence of
appropriate antibiotic treatment, long-term persistence of H.
pylori could contribute to possible gastritis, peptic or duodenal
ulcers, gastric adenocarcinoma, or mucosa-associated lym-
phoid tissue (MALT) lymphoma (Chey et al. 2007;Kuoet
al. 2014; Pritchard and Crabtree 2006). A number of investi-
gations have confirmed that the strong colonization of H.
pylori might give rises to stomach-related malignancies,
resulting in the classification of H. pylori as one of the main
carcinogenic agents (Hagymasi and Tulassay 2014).
While the eradication of H. pylori infection is much of a
concern, attempts for accurate detection of the infection
Farnaz Fahimi and Shamim Sarhaddi contributed equally to this work.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00253-018-9068-4) contains supplementary
material, which is available to authorized users.
* Mohammad Reza Tohidkia
* Yadollah Omidi
Research Center for Pharmaceutical Nanotechnology, Biomedicine
Institute, Tabriz University of Medical Sciences, Tabriz, Iran
School of Advanced Biomedical Sciences, Tabriz University of
Medical Sciences, Tabriz, Iran
Department of Pharmaceutics, Faculty of Pharmacy, Tabriz
University of Medical Sciences, Tabriz, Iran
Department of Microbiology, Faculty of Medicine, Tabriz University
of Medical Sciences, Tabriz, Iran
Applied Microbiology and Biotechnology