The final two inoculations were prepared in 8.0 mL of 0.15 M NaCl containing the respective antigens. The inoculations were performed 15 days apart by subcutaneous injection selleck at four different points of the dorsal region of each animal. Fifteen days after the last inoculation, blood was collected in sterile plastic bags containing anticoagulant solution (citric acid,

1.47 g; sodium citrate, 4.80 g; dextrose, 1.47 g; dissolved in a sufficient amount of distilled water to a final volume of 100 mL) by venipuncture of the jugular vein. The bags were allowed to stand overnight in a refrigerating chamber (4–8 °C). Plasma samples from each horse were pooled and stored at −20 °C. Blood cells resulting from the bleeding were re-infused in the original horse. Four equine plasma samples (Batches No: #143, #158, #223 and #356) and six F(ab′)2 anti-Crotalus

commercial antivenom preparations (Batch #1006140; Batch #100107119; Batch #1007187; Batch #1009230; Batch #1010282; Batch #1010283) were provided by “Divisão de Desenvolvimento Tecnológico e Produção – Seção de Processamento de Plasmas Hiperimunes, Instituto Butantan”. Experimental plasma was obtained by separating plasma from the blood collected from the experimental animals, as described in Section 2.7. The procedure presently used to manufacture horse commercial serum from plasma is completely enclosed MAPK Inhibitor Library solubility dmso and automated (Raw et al., 1996). The procedure used in this study, improved with the introduction of additional filtration and chromatography, included ten steps (Guidolin et al., 2010). Before the antivenom was released to

mafosfamide treat envenomed victims, the purified F(ab′)2 were submitted to a quality control evaluation in order to verify the absence of bacterial contamination, bacterial lipopolysaccharide and toxic substances. The final products were adjusted to contain the desired neutralizing antibody titer in less than 10 mg of protein/ml and were labeled as “Crotalic Antiserum”. One milliliter of the preparation neutralized 1.5 mg of Crotalus venom. Each ampoule contained 10 ml of antivenom. This antivenom, as well as the other antivenoms produced by the “Divisão de Desenvolvimento Tecnológico e Produção – Instituto Butantan”, was prepared according to the recommendations of the World Health Organization (1981). Serum rich in F(ab′)2 fragments was produced as described by Towbin et al. (1979). Western blot analysis was carried out according to the method previously described by Towbin et al. (1979). Crude C. d. terrificus, C. d. collilineatus, C. d. cascavella and C. d. marajoensis venoms (10 μg) and partially purified crotoxin and PLA2 (2 μg) were treated with SDS-PAGE sample buffer under reducing conditions and resolved in a 12.5% polyacrylamide gel. Some preparations were stained with silver sulfate, while others were electroblotted onto nitrocellulose membranes, according the method described by Laemmli (1970).