Zahra Shadman1,2, Safar Farajnia3,4, Mohammad Pazhang2, Mohammadreza Tohidkia5, Leila Rahbarnia6, Saeed Najavand7, Sayna Toraby7. 1. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 2. Department of Cellular and Molecular Biology, Faculty of Science Faculty, Azarbaijan Shahid Madani University, Tabriz, Iran. 3. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. farajnia@gmail.com. 4. Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. farajnia@gmail.com. 5. Nanotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 6. Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 7. Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract
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.
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.
Entities:
Keywords:
Exotoxin A; Human single chain antibody; Phage display; Pseudomonas aeruginosa
Authors: A D Griffiths; S C Williams; O Hartley; I M Tomlinson; P Waterhouse; W L Crosby; R E Kontermann; P T Jones; N M Low; T J Allison Journal: EMBO J Date: 1994-07-15 Impact factor: 11.598
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