Abstract
The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against tumor necrosis factor-alpha (TNF-α), which is a potent pro-inflammatory cytokine and plays important role in various inflammatory diseases and malignancies. To pursue production of scFv antibody fragments against human TNF-α, we performed five rounds of biopanning using stepwise decreased amount of TNF-α (1 to 0.1 μg), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR, restriction fragment length polymorphism (RFLP) and Western blot analyses as well as fluorescence microscopy and flow cytometry. Based upon binding affinity to TNF-α, 15 clones were selected out of 50 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human TNF-α. The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the Raji B lymphoblasts. The effectiveness of the selected scFv fragments was further validated by flow cytometry analysis in the lipopolysaccharide (LPS) treated mouse fibroblast L929 cells. Based upon these findings, we propose the selected fully human anti-TNF-α scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications.
Keywords: Cytokines, immunotherapy, human TNF-α, monoclonal antibody, phage display
Current Pharmaceutical Design
Title:Targeting Cytokines: Production and Characterization of Anti-TNF-α scFvs by Phage Display Technology
Volume: 19 Issue: 15
Author(s): Jalal Abdolalizadeh, Mohammad Nouri, Jafar Majidi Zolbanin, Abolfazl Barzegari, Behzad Baradaran, Jaleh Barar, George Coukos and Yadollah Omidi
Affiliation:
Keywords: Cytokines, immunotherapy, human TNF-α, monoclonal antibody, phage display
Abstract: The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against tumor necrosis factor-alpha (TNF-α), which is a potent pro-inflammatory cytokine and plays important role in various inflammatory diseases and malignancies. To pursue production of scFv antibody fragments against human TNF-α, we performed five rounds of biopanning using stepwise decreased amount of TNF-α (1 to 0.1 μg), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR, restriction fragment length polymorphism (RFLP) and Western blot analyses as well as fluorescence microscopy and flow cytometry. Based upon binding affinity to TNF-α, 15 clones were selected out of 50 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human TNF-α. The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the Raji B lymphoblasts. The effectiveness of the selected scFv fragments was further validated by flow cytometry analysis in the lipopolysaccharide (LPS) treated mouse fibroblast L929 cells. Based upon these findings, we propose the selected fully human anti-TNF-α scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications.
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Abdolalizadeh Jalal, Nouri Mohammad, Majidi Zolbanin Jafar, Barzegari Abolfazl, Baradaran Behzad, Barar Jaleh, Coukos George and Omidi Yadollah, Targeting Cytokines: Production and Characterization of Anti-TNF-α scFvs by Phage Display Technology, Current Pharmaceutical Design 2013; 19 (15) . https://dx.doi.org/10.2174/1381612811319150019
DOI https://dx.doi.org/10.2174/1381612811319150019 |
Print ISSN 1381-6128 |
Publisher Name Bentham Science Publisher |
Online ISSN 1873-4286 |
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