*5.5. Stx2 Neutralization Assay in HGEC*

Neutralization assays were developed according to two procedures: pre-incubation and co-incubation. The pre-incubation was performed by pre-treat HGEC primary cultures with different FabF8:Stx2 concentrations (1 h at 37 ◦C) before exposure of cells to Stx2 for 72 h. On the other hand, for co-incubation, cells were treated simultaneously with FabF8:Stx2 and Stx2 for 72 h. FabF8:Stx2 concentrations used in the experiments ranged from 0.0001 to 10 μg/mL and Stx2 was assayed at the dilution required to kill 50% of cells (1 CD50 = 0.5 ng/mL). The molar ratios used ranged from 0.07:5 to 7000:5 for FabF8:Stx2. Finally, the ability of FabF8:Stx2 to neutralize Stx2 was analyzed by HGEC cell viability as is described below.
