Figure 3

Analysis of the 3F12E7 scFv aggregation state and activity. (a) Evaluation of aggregation propensity of 3F12E7 scFv using Aggrescan3D web server and sequence-based predictors. Amino acid residues (numbered according to primary structure) that had positive scores are indicated in different colors for each predictor: Aggrescan (purple); Aggrescan3D (magenta); Waltz (blue); FoldAmyloid (yellow); Tango (orange). (b) Oligomeric profile of the 3F12E7 scFv, as assessed by blue native gel polyacrylamide electrophoresis (BN-PAGE). Gel was stained with silver nitrate. Graph on the right shows the corresponding densitometry analysis. (c) Dynamic light scattering (DLS) analysis for 3F12E7 scFv (left) and full-length IgG mAb (right). Average particle size results for each mAb are expressed by signal intensity. Each line denotes data obtained for the 3 independent samples. (d) Analysis of 3F12E7 scFv by size-exclusion chromatography (on a PD-10 column) and the binding of the obtained fractions to FGF2, as assessed by ELISA. Dashed line indicates ELISA background signal (BG). (e) Immunoblotting analysis of the 3F12E7 scFv reactivity to B16-F10 tumor protein extracts. For that, antibodies were labeled with biotin and detected with horseradish peroxidase (HRP)-streptavidin. These immunoblot images are also provided in Supplementary Fig. S4b. MW, molecular weight (kDa).