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.