*4.4. UPLC*

Five μL samples (alkylated-ricin, alkylated-RTA or alkylated-RTB) were analyzed with a Water Acquity UPLC (Waters Corporation, Milford, MA, USA) equipped with a UV detector and binary solvent manager. The output signal was monitored and processed using Empower software (Empower 2.0, Waters Corporation, Milford, MA, USA). The method was employed using an Acquity UPLC BEH C-4 1.7 μm (2.1 × 50 mm) column (Waters Corporation, Milford, MA, USA). The flow rate of the mobile phase was 0.4 mL/min. The column temperature was 50 ◦C, and the eluted products were monitored at a wavelength of 215 nm. The samples were rinsed for 4.5 min in an acetonitrile gradient from 70% buffer A (5% acetonitrile in 0.1% trifluoroacetic acid [TFA]) and 30% buffer B (80% acetonitrile in 0.1% TFA), to 30% buffer A.

#### *4.5. Isolation and Purification of Alkylated-Ricin Subunits*

Monomerized ricin preparation was loaded on an α-Lactose-Agarose (Sigma-Aldrich, Rehovot, Israel) column, and extensively washed with PBS for collection of alkylated-RTA. Alkylated-RTB was eluted from the column with 0.5 M galactose. The purity of the isolated subunits was verified by UPLC.

#### *4.6. Assessment of Ricin Activity in a Cell-Free Translational Assay*

The biological activity of ricin was determined in a cell-free assay, as previously described [14,15]. Briefly, rabbit reticulocyte lysate containing luciferase mRNA was used to measure the activity of ricin via inhibition of protein synthesis. The relative biological activity was determined by comparing the luminescence levels in treated samples versus those of untreated controls. The amount of luciferase translated is inversely proportional to the activity of ricin.

#### *4.7. Assessment of Ricin Activity in a Cell Culture*

Genetically engineered HEK-293-acetylcholinesterase (AChE) cells [16] were cultured in Dulbecco's modified Eagle's medium (DMEM) (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal bovine serum (FBS). For the cytotoxicity studies, the cells were seeded in 96-well plates (0.75 × 10<sup>5</sup> cells/well) in medium containing different concentrations of intact or monomerized ricin. Sixteen hours later, the medium was replaced, the cells were incubated for 2 h, and the amount of secreted AChE in each well was assayed according to Ellman et al. [32] in the presence of 0.1 mg/mL bovine serum albumin (BSA), 0.3 mM 5,5'-dithiobis(2-nitrobenzoic acid), 50 mM sodium phosphate buffer (pH 8.0), and 0.5 mM acetylthiocholine iodide (ATC) (Sigma-Aldrich, Rehovot, Israel). The assay was carried out at 27 ◦C and monitored by a Thermomax microplate reader (Molecular Devices, Ramsey, MN, USA).

#### *4.8. In Vitro Ricin Neutralization Assay*

HEK-293-AChE cells [16] were seeded in 96-well plates (0.75 × 10<sup>5</sup> cells/well) in medium containing 2 ng/mL ricin, in the absence or presence of increasing doses of the F(ab')2-based anti-ricin antitoxin. Sixteen hours later, the medium was replaced, the cells were incubated for 2 h, and the amount of secreted AChE in each well was assayed as described above.
