Figure 1

Binding assays of Trastuzumab and Pertuzumab to Her2 on a BLItz system. (a) Whole Trastuzumab binding to Her2. Binding of 12.5–200 nM of Trastuzumab to 25 μg/ml of Ni-NTA probe bound Her2-His-tagged on the BLItz system. The binding kinetics of Trastuzumab was titratable to 25 nM. (b) Whole Pertuzumab binding to Her2. Binding of 12.5–200 nM of Pertuzumab to 25 μg/ml of Ni-NTA probe bound Her2-His-tagged on the BLItz system. The binding kinetics of Pertuzumab was titratable to 25 nM. (c) Whole Trastuzumab binding with Pert preloaded. Binding of 200 nM Trastuzumab to (i) Her2-His-tagged (ii) 50 and 200 nM Pertuzumab or 200 nM human IgG. Unbound sensor was used as negative control. (d) Whole Pertuzumab binding with Trast preloaded. Binding of 200 nM Pertuzumab to (i) Her2-His-tagged (ii) 50 and 200 nM Trastuzumab or 200 nM human IgG. Unbound sensor was used as negative control. (e) Whole Trastuzumab binding with half Pert preloaded. Binding of 200 nM Trastuzumab to (i) Her2-His-tagged (ii) 50 nM Pertuzumab or 200 nM human IgG at 1 min loading time. One minute loading time was used to prevent saturation of Pertuzumab, which may interfere with Trastuzumab binding. (f) F(ab) binding to Her2. Binding assay showing 200 nM of Trastuzumab or Pertuzumab F(ab) to Her2-His-tagged. Decreased maximum binding kinetics reflect the decreased bound protein size. (top left) SDS-PAGE showing purified Trastuzumab and Pertuzumab F(ab) prepared using the Fab preparation kit. (g) Trastuzumab F(ab) binding with Pert F(ab) preloaded. Binding of 200 nM Trastuzumab F(ab) to (i) Her2-His-tagged (ii) 200 nM of Pertuzumab F(ab) or human IgG. (h) Pertuzumab F(ab) binding with Trast F(ab) preloaded. Binding of 200 nM Pertuzumab F(ab) to (i) Her2-His-tagged (ii) 200 nM of Trastuzumab F(ab) or human IgG. Bl, baseline, as measured using phosphate-buffered saline; hIgG, human IgG control; Ni-NTA, Nickel-nitrilotriacetic acid; Pert, Pertuzumab; Trast, Trastuzumab.