*5.2. Antibody Generation and Characterization*

The FabF8:Stx2 was selected by phage display, using a human synthetic antibody library (library F), which displays on the M13 bacteriophage surface a Fab antibody fragment [27]. Selection and panning were performed as described by Sidhu and Fellouse [36] using the protocol of selection against immobilized antigens. In summary, it was used 5 μg/mL toxin (100 μL/well) in phosphate-buffered saline (PBS) to coat a microplate (Maxisorp, Nunc) in the first round and 2.5 μg/mL toxin (100 μL/well) in PBS for the followed selection rounds. The same was performed with the negative protein control (MPB). The coated plate was incubated at room temperature for 2 h or 18 h at 4 ◦C, followed by blocking for 1 h with 200 μL/well PBS-0.2% BSA. Next, the phage library solution in PBT buffer (PBS-0.2% BSA-0.05% Tween-20) was added to the negative control wells (100 μL/well) and the plate was incubated at room temperature for 2 h with gentle shaking. The content of the control wells containing the phage library solution was removed and placed into the toxin-coated wells. Next, the non-binded phages were removed by washing them 10 times with PT buffer (PBS-0.05% Tween-20). Toxin-bound phages were eluted by adding 100 μL/well of 100 mM HCl and incubating at room temperature for 5 min. To neutralize the pH of the eluent, it was transferred to a new 1.5-mL microfuge tube containing 1.0 M Tris-HCl, pH 11. Half of the eluted phage solution was added to 10 volumes of actively growing *E. coli* omnimax (OD600 < 1.0) in 2YT/tet medium, which was then incubated at 37 ◦C for 20 min with shaking at 200 rpm before M13-K07 helper phage were added (10<sup>10</sup> infectious units (IU)/mL) and the whole culture was incubated at 37 ◦C with shaking for an additional hour. The culture was then transferred to a 30 mL 2YT/carb/kan medium, and cells grew at 37 ◦C overnight before phage was harvested for the next round of panning. A serial dilution on LB/carb plates was performed to determine the number of phages eluted and four panning rounds were performed. The phage selected was sequenced and forwarded to cloning and production. The cloning of the Fab expression vector, Fab fragment expression, and purification was performed as previously reported by Luz et al. [25].
