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. 2022 Sep 6;14(1):2115200. doi: 10.1080/19420862.2022.2115200

Figure 2.

Panel A: schematics of yeast cells being selected using equilibrium or kinetic sort. Panel B: selections rounds performed: 3 equilibrium sorts followed by 2 kinetic sorts. Panel C: Flow cytometry analysis of the naïve libraries binding to the antigen. Binding is observed for all libraries at an antigen concentration of 33.3 nM, with stronger binding observed for HCDR1 and 2 libraries. Panel D: Flow cytometry analysis of the last round libraries binding to the antigen. Strong binding was observed for all libraries at an antigen concentration of 0.1 nM, even after 4 h of competition with unlabeled antigen.

(a) Schematic representation of yeast display selections using equilibrium and kinetic protocols. (b) Outline of the selection rounds performed in phase 1 with the three-phase 1 libraries (L1L2, L3, and H1H2). (c) The yeast display binding profiles of the parental scFv and the three starting phase 1 libraries at increasing antigen concentration assessed by flow cytometry. Binding to antigen (APC fluorescence) is shown on the Y-axis and scFv display (PE fluorescence) is shown on the X-axis. (d) Yeast display binding profile of the parental scFv and the three-phase 1 libraries after 5 rounds of selection. A reference gate representing the parental population at the corresponding given concentration is shown. In the last two columns, after labeled antigen incubation, cells were washed and incubated with an excess of unlabeled antigen for 2 h and 4 h to evaluate overall improvement in off-rates.