Abstract
BackgroundPseudomonas 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.MethodsThe 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.ResultsBased 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.ConclusionsThe 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.
Highlights
Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system
Methods Single-chain variable fragment (scFv)-phage library, bacterial strains, and components The semisynthetic human scFv phage libraries I & J (Tomlinson I J), E. coli strains (HB2151 and TG1), and KM13 helper phage were from the Medical Research Council (MRC), Cambridge
Expression and purification of exotoxin A domain I E. coli containing the ExoA-DI encoding construct were cultured in LB media and used for plasmid extraction by FAVORGEN plasmid extraction kit according to the manufacturer instruction
Summary
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. Pseudomonas (p.) aeruginosa is the most common cause of nosocomial infections leading to a high mortality rate, especially in people with cystic fibrosis, neoplastic disease, and severe burns [1]. The second motif (the REDLK-591913–609) is located at the toxin’s carboxylic end and is responsible for retaining the toxin in the endoplasmic reticulum compartment. Both motifs are essential for toxicity [4]. The development of anti-exotoxin A antibody is of great interest for the treatment of pseudomonas infections
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
