*4.1. Ricin Preparation*

Crude ricin was prepared from seeds of endemic *R. communis*, essentially as described before [30]. Briefly, seeds were homogenized in a Waring blender in 5% acetic acid/ PBS. The homogenate was centrifuged and the clarified supernatant containing the toxin was subjected to ammonium sulfate precipitation (60% saturation). The precipitate was dissolved in PBS and dialyzed extensively against the same buffer. The toxin preparation appeared on a Coomassie Blue stained non-reducing 10% polyacrylamide gel as 2 major bands of molecular weight approximately 65 kDa (=ricin toxin, ~80%) and 120 kDa (=ricinus communis agglutinin (RCA), ~20%). Pure toxin was prepared as described previously [30,31]. Briefly, under laminar flow and aseptic conditions the crude ricin preparation was loaded onto a gel-filtration column (Superdex 200HR 16/60 Hiload prep grade on an AKTA explorer, GE Healthcare Bio-Science AB, Uppsala, Sweden) and washed out with PBS to yield two protein peaks, corresponding to RCA and ricin. The purity of the ricin fraction was estimated by SDS-PAGE analysis to be >98%. Protein concentration was determined by 280 nm absorption (Nanodrop).

#### *4.2. Reduction and Alkylation of Pure Ricin*

Monomerized ricin vaccine was prepared, essentially as previously described [13]. Briefly, pure ricin was incubated with 50 mM Dithiothreitol (DTT, Sigma-Aldrich, Rehovot, Israel) for 2 h in room temperature, the ricin-DTT solution was incubated for additional 2 h (room temperature, protected from light) with 100 mM Iodoacetamide (IAA, Sigma-Aldrich, Rehovot, Israel), and the product (alkylated-ricin) was extensively dialyzed against PBS.
