Fig. 3
Binding analysis of purified scFvs. A Gel filtration chromatography profile of AU1. Monomer forms of scFvs were eluted at around 75 ml. Inset is the 12 % SDS-PAGE profile with purified scFv. B Direct ELISA with four selected purified scFvs against BSA conjugated U1 peptide with two-fold serial dilutions. Only scFv UU1 showed weak binding with peptide compared to other scFvs. C Bio-layer interferometry was carried out with all scFvs at different concentrations. Colors represent different concentrations that were serially diluted up to three times. All scFvs have a binding affinity (KD) in the sub-micromolar range. D Stable expression of native M2 protein on the surface of HEK293T cells was carried out, and its binding with the scFvs was performed by flow cytometry analysis. All scFvs showed a positive shift as compared to the control. The graph represents the average mean of the peaks. All experiments were performed in triplicate. Statistical analysis was performed using the one-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparisons. ** P < 0.01; * P < 0.05. Data points represent averages of triplicates, and error bars represent standard deviations