Anti-INS Recombinant Antibody (Clone 5ins16) (CAT#: PABS-0026)

Figure 1 Ligand binding

Antibody affinity was first determined by yeast display titrations (■), and then improved through random mutagenesis and in vitro selection using FACS, yielding affinity improvement from an apparent Kd = 300 to 30 nM in 5ins16 (□).

Baran, D., Pszolla, M. G., Lapidoth, G. D., Norn, C., Dym, O., Unger, T., ... & Fleishman, S. J. (2017). Principles for computational design of binding antibodies. Proceedings of the National Academy of Sciences, 114(41), 10900-10905.

Figure 2 Surface plasmon resonance traces.

The antibodies were expressed as Fab- and aminecoupled to CM5 chips. The affinities determined from the kinetic fits were 50 nM for 5ins16.

Baran, D., Pszolla, M. G., Lapidoth, G. D., Norn, C., Dym, O., Unger, T., ... & Fleishman, S. J. (2017). Principles for computational design of binding antibodies. Proceedings of the National Academy of Sciences, 114(41), 10900-10905.

Figure 3 Mutation analysis of designed binding modes.

Relative binding signal of variants containing single-point mutations in ligand-binding sites (gray bars) or outside the ligand-binding sites (black bars). Additional manual mutations were introduced on the ligand ACP (cyan spheres) in the intended binding surface (gray bars) or outside the binding surface (black bars) and tested for binding of the affinitymatured antibodies. Finally, mutations were introduced in the designed antibodies also through random mutagenesis and in vitro selection of improved binders (blue spheres). Mutations at the binding surface reduced affinity, except for Val71Glu on ACP, which enhanced binding of the affinity-matured 2acp12_ev. Mutations away from the interface, including those isolated from in vitro selection, increased binding affinity. Binding signals were tested in yeast surface display with the antibodies expressed as scFv and the ligand at 50 nM concentration for 2acp12_ev and at 1 μM concentration for 2acp12, 5acp14_ev, and 5ins16.

Baran, D., Pszolla, M. G., Lapidoth, G. D., Norn, C., Dym, O., Unger, T., ... & Fleishman, S. J. (2017). Principles for computational design of binding antibodies. Proceedings of the National Academy of Sciences, 114(41), 10900-10905.