ELISA was used with the aim of evaluating the antigenic cross-reactivity

of S. plumieri whole venom with Stonefish antivenom. The assays were performed as described previously by Chávez-Olórtegui PI3K activation et al., 1991. Falcon flexible microtitration plates purchased from Becton Dickinson Labware Europe (Becton Dickinson France S.A.) were coated with 100 μl of a 5 μg/ml solution of the S. plumieri venom in 0.02 M sodium bicarbonate buffer, pH 9.6 and incubated overnight at 5 °C. After blocking non-specific sites with 2% (w/v) casein solution for 1h at 37 °C, the immobilized venom proteins were titrated with decreasing concentrations of stonefish antivenom (from 1:200 to 1:204800 dilution) and incubated at 37 °C for 1h.

Non-specific binding was measured in the presence of pre-immune horse serum at the same conditions. Bound IgG was detected via peroxidase conjugated antibody raised against Selleck MG 132 horse IgG diluted 1:1000. Wells coated with 2% casein were taken as blank and subtracted from all values. Absorbance values were determined at 492 nm with a Titertek Multiscan spectrophotometer. All measurements were made in triplicate and the results expressed as the mean of two assays. Results were expressed as mean ± SEM (Standard Error of the Mean) and were evaluated using one- or two-way analysis of variance (ANOVA) followed by the Tukey post hoc test. Results were also evaluated by Student’s t-test. In all cases, differences were considered significant at p < 0.05. For determination of the edematogenic response induced by S. plumieri venom, doses of 7.5, 15 and 60 μg of venom/animal were used. Fig. 1A shows the time-course evaluation of edematogenic

effect. It is possible to observe that the venom induced an intense and sustained dose-dependent edematogenic response with a maximal activity observed 30 min after injection of 58 ± 6% with 7.5 μg, 61 ± 6% with 15 μg, and 82 ± 2% check with 60 μg of protein/animal. The edema remained significantly elevated compared to control group over 6 h at the dose of 7.5 μg, 24 h at the dose of 15 μg and 72 h at the dose of 60 μg. Higher doses were unable to increase the edematogenic response compared to the response induced by 60 μg of SpV (data not shown). Likewise, a significant nociceptive response was observed. Fig. 1B shows that the SpV induced an increase of paw licking duration that reached its maximum with 15 μg of protein/animal (124.5 ± 29.3 s). Doses >15 μg of S. plumieri venom were unable to increase the paw licking duration in a dose-related way, nevertheless each dose presented significant values ( Fig. 1B). The vehicle control (PBS) had no significant effect on the experiment. The ability of SFAV in neutralizing the inflammatory activity induced by S. plumieri venom was evaluated by pre-incubation of SpV with SFAV. Fig. 2 shows that SFVA succeeded in neutralizing the in vivo edematogenic and nociceptive effects of SpV.