2.3 | Phage panning, mAb-EspB-B7 expression and purification
A human synthetic-phage library, displaying single-chain variable fragment (scFv), was used to isolate antibodies targeting EspB as described previously (Azriel-Rosenfeld, Valensi, & Benhar, 2004). mAb-EspB-B7 VH and VL were cloned in mammalian expression vectors (pcDNA3.4H and pcDNA3.4L encoding the IgG1 heavy and lambda light chain constant regions) by Gibson cloning. The cloned vectors were transformed into E. coli competent cells (XL-1 blue) and were purified using plasmid purification kit (Invitrogen). The vectors were co-transfected into Expi293 expression system (Gibco) according to the manufacturer’s instructions. Transfected Expi293 cells were harvested by centrifugation at 2000 × g for 10 min at 4°C and conditioned medium was applied to MabSelect affinity column (GE Healthcare) according to the manufacturer’s instructions.