title: Improved Soluble ScFv ELISA Screening Approach for Antibody Discovery Using Phage Display Technology.
abstract: Phage display technology (PDT) is a powerful tool for the isolation of recombinant antibody (Ab) fragments. Using PDT, target molecule-specific phage-Ab clones are enriched through the "biopanning" process. The individual specific binders are screened by the monoclonal scFv enzyme-linked immunosorbent assay (ELISA) that may associate with inevitable false-negative results. Thus, in this study, three strategies were investigated for optimization of the scFvs screening using Tomlinson I and J libraries, including (1) optimizing the expression of functional scFvs, (2) improving the sensitivity of ELISA, and (3) preparing different samples containing scFvs. The expression of all scFv Abs was significantly enhanced when scFv clones were cultivated in the terrific broth (TB) medium at the optimum temperature of 30 °C. The protein A-conjugated with horseradish peroxidase (HRP) was found to be a well-suited reagent for the detection of Ag-bound scFvs in comparison with either anti-c-myc Ab or the mixing procedure. Based on our findings, it seems there is no universal media supplement for an improved expression of all scFvs derived from both Tomlinson I and J libraries. We thus propose that expression of scFv fragments in a microplate scale is largely dependent on a variety of parameters, in particular the scFv clones and relevant sequences.
authors.affiliation: 1 Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Mohammad R
authors.initials: MR
authors.lastname: Tohidkia
authors.affiliation: 1 Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.|2 Department of Biochemistry, Higher Education Institute of Rab-Rashid, Tabriz, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Sepehri
authors.affiliation: 1 Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Shirin
authors.initials: S
authors.lastname: Khajeh
authors.affiliation: 1 Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.|3 Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Jaleh
authors.initials: J
authors.lastname: Barar
authors.affiliation: 1 Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.|3 Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Yadollah
authors.initials: Y
authors.lastname: Omidi
mesh_terms: D003470:Culture Media; D055808:Drug Discovery; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000032:analysis*; D012995:Solubility; D013696:Temperature
chemical_list: D003470:Culture Media; D019151:Peptide Library; D057127:Single-Chain Antibodies
keywords: antibody; biopanning; phage display; scFv screening