He2 h822r

Plates were coated overnight at 4°C with 1.0 μg/mL anti-CD19 antibody FMC63 (#NBP2-52716, Novus Biologics, Centennial, CO) or 1.0 μg/mL purified Her2-Fc (#HE2-H5253, AcroBiosytems, Newark, DE), in 0.1 M carbonate, pH 9.5. The plate was blocked with 0.3% non-fat milk in TBS for 1 hour at room temperature. After washing in TBST 3 times, the bridging protein was titrated from a starting concentration of 5 μg/mL using serial 3-fold dilutions in TBS/1% BSA and incubated 1 hour at room temperature. Detection with Her2-Fc, biotinylated Her2 (#He2-H822R, AcroBiosystems), or antibody FMC63 was performed by adding the detection reagent at 1.0 μg/mL and incubating for 1 hour at room temperature. The plates were then washed 3 times, followed by a 1 hour incubation with a 1:2000 dilution of anti-human IgG-HRP (#109-035-088, Jackson ImmunoResearch, West Grove, PA) for Her2-Fc detection, HRP-steptavidin (#21130, Thermo Fisher) for biotinylated Her2 detection, and anti-mouse IgG-HRP (#115-035-062, Jackson ImmunoResearch) for FMC63 detection. Then, 1-Step Ultra TMB-ELISA solution (#34028, Thermo Fisher) was added to develop the peroxidase signal, and the plate was read at 405 nm. Curves were fit using a four parameter logistic regression to calculate the EC₅₀.

Kinetics of binding for all anti-HER2 Fabs was measured using Octet BLI and SA-coated tips (18-5136; Sartorius). In all cases, the buffer used was DPBS (14190-169; Gibco) with 0.1% BSA and 0.02% Tween 20. Purified Fabs were diluted to a final concentration of 20 nM. Kinetics were measured using the following steps: (1) sensor check for 60 seconds, (2) loading of human HER2–biotin (HE2-H822R, 25 µg; Acro Biosystems) at 5 µg ml⁻¹ for 30 seconds, (3) baseline measurement for 60 seconds, (4) association kinetics at 20 nM of each Fab for 300 seconds, and (5) dissociation kinetics for 600 seconds in buffer.