Target binding activity of HB-002.1. Target binding activity of intact as well as deglycosylated HB-002.1 was analyzed by ELISA. (A) Binding to VEGF-A was compared to bevacizumab and Flt1(2). hIgG-Fc was used as negative control. (B-C) Binding to VEGF-B (B) and PIGF (C) was compared with rhVEGFR1-Fc. (D) Kinetic binding affinity was measured by equilibrium binding assays that measures unbound HB-002.1 or bevacizumab after incubation of 0.5 nM of HB-002.1 or bevacizumab with varying amounts of VEGF-165. (E) HB-002.1 deglycosylated by treatment with N-glycosidase F (D) or not deglycosylated (ND) was separated by SDS-PAGE under reducing or non-reducing conditions and visualized by staining with Coomassie Brilliant Blue. (F) VEGF-A binding affinity was compared between intact (non-digested) and deglycosylated (digested) HB-002.1.