The horses were subjected to at least two cycles of re-immunizations. All doses were diluted in sterile

saline buffer. The horses were bled one week after the last injection. Approximately 50 ml of blood was collected and subjected to hemosedimentation at 37 °C for 1 h and the supernatant was centrifuged. The fraction obtained (anti-Loxosceles serum) was stored at −20 °C. Forty-eight New Zealand rabbits were used to assess the neutralizing potency of the ten horse sera used in this study. The neutralizing capacity of the anti-Loxosceles sera was assessed using the methodology described by Furlanetto (1961). The quantity of the venom that was inoculated into the rabbits was based on the minimum necrotic dosage (MND) as reported by Theakston et al. (2003). For this test, only the venom from L. intermedia was inoculated (intradermally) on the inner ear of a rabbit (3 BMS354825 animals/dilution of sera tested) with 1 ml of intravenous injection (marginal vein of the opposite ear) of serum (1:8 and/or 1:6 dilutions) in saline buffer. Initially, the serum dilution was 1:8 and the animals were observed for 72 h for the appearance of necrosis. During this time period, the appearance of necrosis indicated that the diluted horse serum was not sufficient

to neutralize the venom. In that case, a 1:6 serum dilution was then used. Sera, which were not able to neutralize 6 MND of the venom, were considered to be of low neutralizing potency. ELISA was performed by coating plates (Falcon, see more Becton Dickinson) overnight

at 4 °C with 100 μl of a 2.5 μg/ml solution of crude venom from the three species of Loxosceles (L. intermedia, L. gaucho, and L. laeta) in carbonate buffer (0.05 mM) at pH 9.6. After washing and blocking (with 2% casein for 1 h at 37 °C) the plates, they were incubated in diluted sera (1:1000; 1:5000; 1:20 000; and 1:40 000) under the same conditions. Peroxidase-conjugated anti-horse IgG antibody (Sigma, 1:30 000) was added and the plates were incubated for 1 h at 37 °C. After rinsing the plates, a substrate (citrate buffer pH 5.0, hydrogen peroxide, and ortho-phenyldiamine) was added. The reaction was stopped by adding 20 μl of 5% H2SO4; the antibody NADPH-cytochrome-c2 reductase reactivity was determined by the intensity of the staining. Absorbance values were determined at 492 nm using an ELISA Bio-Rad 550. All measurements were done in duplicate and the results were expressed as the mean of three assays. Different ELISA conditions were tested: the composition of the synthetic peptides (Pep1-BSA, Pep2-BSA, Pep3-BSA, Pep1-BSA + Pep2-BSA, Pep1-BSA + Pep3-BSA, Pep2-BSA + Pep3-BSA, or Pep1-BSA + Pep2-BSA + Pep3-BSA), the antigen concentration (2.5, 5.0, 25.0, 50.0, or 100.0 μg/ml), the plate coating, and the sera dilution (1:1000 and 1:10 000). All measurements were done in triplicate. Two hundred seventy-eight overlapping pentadecapeptides (15-mer) frame-shifted by 3 residues covering the amino acid sequence of the SMase-D proteins of L.