FabF8:Stx2 Characterization

*Surface Plasmon Resonance*—The antibody affinity was determined by surface plasmon resonance (BIAcore T200, Cytiva, Little Chalfont, UK) following the manufacturer's rec-

ommendations. The experiments used HBS-EP buffer, pH 7.4, containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20 as the running buffer. Briefly, Stx2 (purchased from Tufts University School of Medicine, Boston, MA, USA) at 5 μg/mL in 10 mM sodium acetate buffer, pH 5.5 was immobilized (152 RU) on CM5 sensor chips activated by mixing equal amounts of *N*-ethyl-*N*'-(dimethyl aminopropyl) carbodiimide (EDC) and *N*-hydroxysuccinimide (NHS) following the standard immobilization protocol. The sample preparation was in HBS-EP buffer (0–7.4 μM, twofold dilutions) and the kinetic study was performed by a multicycle model at 25 ◦C and a flow rate of 30 μL/min (contact of 120 s and dissociation of 600 s). The sensor chip was regenerated between cycles by a 15 μL pulse of 100 mM glycine containing 2 mM MgCl2, pH 2. The kinetic affinity constant (KD) was calculated using BIAevaluation version 3.0, using the Langmuir 1:1 binding model. Stx2 monoclonal antibody was employed as a control [36]. The experiments were performed in duplicate.

*EC*<sup>50</sup> *definition*—Half-maximal effective concentration (EC50) was performed as described by Luz et al. [25] by coating a 384-well plate (Maxisorp) with 2 μg/well of antigen and incubating overnight at 4 ◦C with gentle shaking, followed by blocking step with 0.2% PB buffer for 1 h at room temperature with gentle shaking. A log 3 serial Fab/scFv dilutions, starting with 20 μg/mL, were performed in PBT, and incubated for 30 min at room temperature with gentle shaking. The assay development was performed after 30 min incubation with gentle shaking using HRP antibody/anti-Flag conjugated to peroxidase (1:5000) in PBT followed by addition of TMB (1:1) and stop with 1 M H3PO4. Several washes with PBT were performed between each incubation. The plate was read with a 450 nm filter. Specificity and absence of cross-reactivity were performed by ELISA as described by Luz et al. [25] using a 96-well plate (Maxisorp) coated with different concentration Stx2 or Stx1 purified toxins (5 μg/mL) incubated 18 h at 4 ◦C with gentle shaking, followed by blocking with 0.2% PB buffer for 1 h at room temperature with gentle shaking. The EC50 concentration of FabF8:Stx2 was added to the plate and incubated for 30 min at room temperature with gentle shaking, followed by 8 times washing with PT. Next, it was added (100 μL/well) into the wells, HRP antibody/anti-Flag conjugated to peroxidase (1:5000) in PBT, which was then incubated for 30 min at room temperature with gentle shaking. Again, the plate was washed 8 times with PT. The reaction was developed by adding 30 μL/well of TMB (1:1) and stopped by adding 30 μL/well of 1 M H3PO4, and the plate was read with a 450 nm filter.

#### *5.3. Vero Cell Antibody Neutralization Assay*

The certified Vero cell lineage was purchased from Instituto Adolfo Lutz (São Paulo, SP, Brazil). The STEC bacterial supernatant was obtained as described by Shiga et al. [37]. Vero cells (1 × <sup>10</sup><sup>5</sup> cells/mL) were grown in 96-well plates in Dulbecco's medium (DMEM) supplemented with 10% FBS and 30 μg/mL gentamicin, at 37 ◦C in a 5% CO2 atmosphere, for 24 h. The FabF8:Stx2 neutralizing ability was determined by pre incubating for 2 h the Stx2 toxin at the CD50, (i.e., 0.5 μg/mL) defined by Rocha et al. [38] or bacterial supernatants (diluted 1:50) with the same volume of an EC50 concentration of Fab diluted in DMEM supplemented with 2% of FBS at 37 ◦C for 72 h with 5% CO2. After incubation, the viable cells were accessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St Louis, MO, USA) as described by the manufacturer's instructions. These assays were performed three times in duplicate.
