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
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