Ali Akbar Alizadeh1, Maryam Hamzeh-Mivehroud2, Siavoush Dastmalchi3. 1. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Students Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran. ; School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 2. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 3. Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract
PURPOSE: Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Because of central role of TNF-α in pathogenesis of inflammatory diseases, in the current study, we aimed to identify novel scFv antibodies against TNF-α using phage display technology. METHODS: Using libraries composed of phagemid displaying scFv antibodies, four rounds of biopanning against TNF-α were carried out, which led to identification of scFvs capable of binding to TNF-α. The scFv antibody with appropriate binding affinity towards TNF-α, was amplified and used in ELISA experiment. RESULTS: Titration of phage achieved from different rounds of biopanning showed an enrichment of specific anti-TNF-α phages during biopanning process. Using ELISA experiment, a binding constant (Kd) of 1.11 ± 0.32 nM was determined for the phage displaying J48 scFv antibody. CONCLUSION: The findings in the current work revealed that the identified novel scFv antibody displayed at the N-terminal of minor coat proteins of phagemid binds TNF-α with suitable affinity. However, the soluble form of the antibody is needed to be produced and evaluated in more details regarding its binding properties to TNF-α.
PURPOSE:Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological and pathological pathways. Because of central role of TNF-α in pathogenesis of inflammatory diseases, in the current study, we aimed to identify novel scFv antibodies against TNF-α using phage display technology. METHODS: Using libraries composed of phagemid displaying scFv antibodies, four rounds of biopanning against TNF-α were carried out, which led to identification of scFvs capable of binding to TNF-α. The scFv antibody with appropriate binding affinity towards TNF-α, was amplified and used in ELISA experiment. RESULTS: Titration of phage achieved from different rounds of biopanning showed an enrichment of specific anti-TNF-α phages during biopanning process. Using ELISA experiment, a binding constant (Kd) of 1.11 ± 0.32 nM was determined for the phage displaying J48 scFv antibody. CONCLUSION: The findings in the current work revealed that the identified novel scFv antibody displayed at the N-terminal of minor coat proteins of phagemid binds TNF-α with suitable affinity. However, the soluble form of the antibody is needed to be produced and evaluated in more details regarding its binding properties to TNF-α.
Entities:
Keywords:
Antibody library; Phage display; Single chain variable fragment; TNF-α
Authors: H J de Haard; N van Neer; A Reurs; S E Hufton; R C Roovers; P Henderikx; A P de Bruïne; J W Arends; H R Hoogenboom Journal: J Biol Chem Date: 1999-06-25 Impact factor: 5.157
Authors: Jennifer Huen; Zhun Yan; Jeremy Iwashkiw; Shraddha Dubey; Maria C Gimenez; Maria E Ortiz; Saumil V Patel; Michael D Jones; Ali Riazi; Mauricio Terebiznik; Saeid Babaei; Dea Shahinas Journal: Front Microbiol Date: 2019-11-26 Impact factor: 5.640