*4.9. Animal Studies*

Animal experiments were performed in accordance with the Israeli law and were approved by the Ethics Committee for animal experiments at the Israel Institute for Biological Research (Horse protocol identification code: H-01-2015, date of approval: 9 August 2015; Rabbits protocol identification codes: RB-20-2011, RB-36-2012, dates of approval: 30 August 2011, 20 November 2012, respectively; Mice protocol identification code: M-59-2016, date of approval: 8 September 2016). Treatment of animals was in accordance with regulations outlined in the USDA Animal Welfare Act and the conditions specified in the Guide for Care and Use of Laboratory Animals (National Institute of Health, 1996). Local horse was immunized to produce the antitoxin preparation. New Zealand white rabbits (Charles River Laboratories Ltd., Canterbury, UK) weighing 2.5 to 3 kg were immunized in order to produce rabbit-derived polyclonal anti-ricin antibodies. Female CD-1 mice (Charles River Laboratories Ltd., UK) weighing 27–32 g were used for toxicity, survival and pathogenesis studies.

Prior to all studies in mice and rabbits, the animals were habituated to the experimental animal unit for 5 days. All mice were housed in filter-top cages in an environmentally controlled room and maintained at 21 ± 2 ◦C and 55 ± 10% humidity. Lighting was set to mimic a 12/12 h dawn to dusk cycle. Animals had access to food and water ad libitum.

#### *4.10. Rabbit Anti-Ricin Hyperimmune Serum Production*

Rabbits were immunized with native- or reduced- ricin in a stepwise manner, injections 1, 2, 3, and 4 containing 0.5, 4, 16, and 100 μg toxin/rabbit, with 4-week intervals between injections. Blood samples were collected (1 week after injection) to ascertain anti-ricin antibody titer build-up. Immunization was continued over 16 weeks, until steady high anti-ricin titers were observed.

## *4.11. Safety Studies in Mice*

Mice were injected intraperitoneally with 40 μg/kg monomerized ricin, at a volume of 200 μL. Mice body weights were monitored for 14 days. In addition, careful individual detailed clinical examinations were carried out. The observations included, among others, changes in skin, fur, eyes, and occurrence of secretions and excretions. Changes in posture and autonomic activity (lacrimation and piloerection) and the presence of bizarre behavior were also checked.

#### *4.12. In Vivo Ricin Neutralization Determination*

Mice were injected intramuscularly with different volumes of rabbit-derived anti-ricin antisera, and 24 h later, the mice were intramuscularly intoxicated with a lethal dose of ricin. Preceding these studies, we determined that 15 μg crude ricin/kg body weight is approximately equivalent to 1 mouse (intramuscular) LD50. Mortality was monitored over 14 days.

## *4.13. Survival Experiments in Mice*

For intranasal intoxication, mice were anesthetized by an intraperitoneal (i.p.) injection of ketamine (1.9 mg/mouse, Vetoquinol, Lure, France) and xylazine (0.19 mg/mouse, Eurovet Animal Health, AD Bladel, The Netherlands). Crude ricin (50 μL; 7 μg/kg diluted in PBS) was applied intranasally (2 × 25 μL) and mortality was monitored over 14 days. Preceding these studies, we determined that 3.5 μg crude ricin/kg body weight is approximately equivalent to 1 mouse (intranasal) LD50. Treatments via the intranasal route were performed on mice anesthetized as above. For antibody treatment following intranasal exposure to ricin, anti-ricin antitoxin was delivered intranasally or intravenously at 24 or 48 h following intoxication.

#### *4.14. Bronchoalveolar Lavage Fluid (BALF) Analysis*

Mice BALF were collected by instillation of 1 mL PBS at room temperature and were centrifuged at 3000 rpm at 4 ◦C for 10 min. Supernatants were collected and stored at −20 ◦C until further use.

BALF levels of IL-6 were determined by ELISA (R&D systems, Minneapolis, MN, USA). Cholinesterase (ChE) enzymatic activity was measured according to Ellman et al. [32]. Assays were performed in the presence of 0.5 mM acetylthiocholine (Sigma-Aldrich, Rehovot, Israel), 50 mM sodium phosphate buffer pH 8.0 (Sigma-Aldrich, Rehovot, Israel), 0.1 mg/mL BSA (Sigma-Aldrich, Rehovot, Israel), and 0.3 mM 5,5-dithiobis-(2-nitrobenzoic acid) (Sigma-Aldrich, Rehovot, Israel). The assay was carried out at 27 ◦C and monitored by a Thermomax microplate reader (Molecular Devices, Ramsey, MN, USA). Protein levels in BALF were determined by 280 nm absorption (NanoDrop 2000, ThermoFisher Scientific, Waltham, MA, USA). Xanthine oxidase (XO) in BALF was determined by an activity assay kit (Molecular Probes, Eugene, OR, USA).

## *4.15. Horse Vaccination and Plasmapheresis*

A horse was immunized with escalating doses of the monomerized ricin until a minimal level of neutralizing antibodies titer is elicited. The first three doses were 2, 5 and 10 milligrams in Incomplete Freund's adjuvant (Statens Serum Institute, Copenhagen, Denmark). The following doses were adjuvant-free, at doses of 10 mg. Plasmapheresis of the hyperimmunized horse was conducted every three months using veterinary plasmapheresis instrument (plasma collection system, PCS-2, Haemonetics Corporation, Braintree, MA, USA). Ten liters of plasma were collected during each plasmapheresis procedure and plasma bags were stored at −20 ◦C.
