Figure 3.

Screening strategy for enrichment of pH-dependent antibody variants from the yeast displayed (A) VH-library and (B) VL-library using FACS. Yeast cells were incubated with unlabeled rhTNF, transferred to PBS-2 (pH 6.0), followed by double staining with directly or indirectly labeled rhTNF and PE or FITC conjugated anti-Penta-His for simultaneous detection of antigen binding and surface display (upper panels, rounds 1, 2, 3a and 3b). After round 2, library cells were subjected to 2 parallel selections. In round 3, incubation with PBS-2 was done either for 30 min (round 3a) or for 10 min (round 3b). Cells in the sorting gates were isolated, grown and subjected to the next round of selection. Percentages of cells in the gates are indicated. Library displaying cells that served as gating controls were incubated with PBS-1 (pH 7.4) instead of PBS-2 and are shown in the lower panel.