Figure 5.

(a and b) Thermal challenge DELFIA assay demonstrates clones with improved thermal stability following phage display optimization. Cook-and-bind DELFIA time resolved fluorescence (TRF) measuring binding to human GUCY2C protein at 60°C, and 65°C, (X-axis) were compared with normal binding with no thermal challenge (Y-axis). Clones from individual CDR soft-randomization libraries are shown in green. Clones from CDR shuffling are shown in blue. The parental clone is shown in red. (c) ScFv-Fc binding activity following incubation at a range of temperatures, from which a T₅₀ can be derived. Parental clone GUCY2C-0295 as scFv-Fc is shown in red and other representative clones with a range of T₅₀ values are shown in orange, purple, blue and green. (d) DSC profiles show a spectrum of unfolding over a range of temperatures, from which a T_m1 can be derived. Representative clones are highlighted with the same coloring. (e) Correlation between T₅₀ and T_m1 using a linear regression model