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. 2015 Apr 7;290(21):13017–13027. doi: 10.1074/jbc.M115.650903

FIGURE 2.

FIGURE 2.

Affinity maturation of hu3F8 scFv by random mutagenesis. A, FACS for yeast display selection. The yeast library was labeled with mouse anti-c-Myc antibody followed by goat anti-mouse dye as well as biotinylated GD2 followed by streptavidin dye. During three FACS selections, yeast cells were stained with concentrations of biotinylated GD2 at 100 μm (Sort 1), 100 μm (Sort 2), and 25 μm (Sort 3), respectively. The 0.1–0.3% cells were selected from sort gates. B, ELISA of yeast-selected hu3F8 scFv variants against GD2. Three scFvs (S4.6, S4.10, and S4.13) and parental hu3F8 scFv were serially diluted and bound with GD2, followed by measurement as optical densities (OD) at 490 nm. C, comparative binding kinetics of yeast-selected hu3F8 scFv variants on solid phase GD2 when measured by surface plasmon resonance. An overlay plot of association and dissociation curves was obtained against immobilized GD2. RU, resonance units.