(A) Binding curves and measurements inferred from Tite-Seq data. (B) Mean fluorescence values (dots) and corresponding inferred binding curves (lines) obtained by flow cytometry measurements for five selected scFvs (WT, OPT, C5, C45, and C107). In (A,B), values corresponding to 0 M fluorescein are plotted on the left-most edge of the plot, dotted lines show the upper ( M) and lower ( M) limits on sensitivity, vertical lines show inferred values, and different shades correspond to different replicate experiments. (C) Comparison of the Tite-Seq-measured and flow-cytometry-measured values for all clones tested. Colors indicate different scFv protein sequences as follows: WT (purple), OPT (green), (black), 1H clones (blue), and 3H clones (red). Each value indicates the mean value obtained across all replicates, with error bars indicating standard error. Clones with outside of the affinity range are drawn on the boundaries of this range, which are indicated with dotted lines. The coefficient of determination () between log Tite-Seq values and log flow values includes clones outside of the affinity range; in such cases, the corresponding boundary value ( M or M) has been used. The amino acid sequences and measured values for all clones tested are provided in Table 1. Figure 4—figure supplement 1 provides plots, analogous to panels A and B, for all of the assayed clones. Figure 4—figure supplement 2 compares and values obtained across all three Tite-Seq replicates. Figure 4—figure supplement 3 quantifies measurement error using synonymous mutants. Figure 4—figure supplement 4 provides information about library composition. Figure 4—figure supplement 5 illustrates the poor correlation between scFv enrichment and Tite-seq measured values. Figure 4—figure supplement 6 shows a 2-fold difference in the specific activities of OPT and WT scFvs. Figure 4—figure supplement 7 illustrates the simulations we used in Figure 4—figure supplement 8 to validate the ability of our analysis to infer correct values.