Isolation and characterizations of a novel recombinant scFv antibody against exotoxin A of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library. METHODS: The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot. RESULTS: Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A. CONCLUSIONS: The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.

Selection of a fully human single domain antibody specific to Helicobacter pylori urease.

Helicobacter pylori bacteria are involved in gastroduodenal disorders, including gastric adenocarcinoma. Since the current therapies encounter with some significant shortcomings, much attention has been paid to the development of new alternative diagnostic and treatment modalities such as immunomedicines to target H. pylori. Having used phage display technology, we isolated fully humane small antibody (Ab) fragment (VL) against the Flap region of urease enzyme of H. pylori to suppress its enzymatic activity. Solution biopanning (SPB) and screening process against a customized biotinylated peptide corresponding to the enzyme Flap region resulted in the selection of VL single domain Abs confirmed by the enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting. The selected Ab fragments showed a high affinity with a KD value of 97.8 × 10-9 and specificity to the enzyme with high inhibitory impact. For the first time, a VL single domain Ab was isolated by SPB process against a critical segment of H. pylori urease using a diverse semi-synthetic library. Based on our findings, the selected VL Ab fragments can be used for the diagnosis, imaging, targeting, and/or immunotherapy of H. pylori. Further, Flap region shows great potential for vaccine therapy.