Figure 10.

Combinatorial bsAb antibody screening. Several bsAbs B10v5xhu225L, B10v5xhu225H, F06xhu225H, anti-CD40 IgG and C5xC6 have been reconstituted in plate format as described before, to prove suitability as a screening platform based on higher and lower affinity profiles. (a) Reconstituted bsAbs were analyzed by ELISA and detected after binding to corresponding antigens with POD-labeled anti-human Fc secondary antibody. The 4–7 bars in Figure 9a represent replicants for reconstitution reproducibility. (b) Functionality of reconstituted c-METxEGFR bsAbs was demonstrated by inhibition of c-MET and EGFR phosphorylation. A549 cells with high to moderate c-MET and EGFR expression levels were incubated with 300 nM bsAb after starvation and treatment with and without 100 ng mL⁻¹ EGF and HGF. Phosphorylated c-MET and EGFR was detected by specific AP-labeled phospho-c-MET or phospho-EGFR secondary antibodies after cell lysis with RIPA buffer followed by SDS-PAGE and western blot analysis. Cetuximab was used as a positive control for EGFR phosphorylation inhibition, while anti-HEL was used as a negative isotype control.