9%. Two hours later, mice were sacrificed by cervical dislocation and back skin was totally removed in order to measure the area of the hemorrhagic lesion. MHD was defined by the dose causing a lesion with a diameter of 10 mm. PLA2 activity was measured using an indirect hemolytic assay (Gutierrez et al., 1988). Increasing concentrations of B. andianus venom (from 0.004 μg up to 10 μg) were prepared in a final volume of 15 μl in PBS and added to 2 mm wells in agarose gel plates (0.8% in PBS, pH = 8.1, containing 1.2% sheep erythrocytes, 1.2% egg yolk and 100 mM CaCl2). Plates were incubated at 37 °C for 18 h and the diameters of the hemolytic haloes were measured.

In controls, 15 μl of PABA was used. One unit (Minimum PLA2 Doses-MPD) corresponds to a minor concentration Dasatinib mw of venom which produced a hemolytic halo of 10 mm diameter. Experiments

were conducted in triplicate. Proteolytic high throughput screening activity was measured with dimethylcasein (Sigma) as described in Lin et al. (1969) with the modifications described in Sanchez et al. (2000). Dilutions corresponding to 5, 10, 20 and 40 μg of venom were used and absorbance values were determined at 340 nm. One unit was defined as ΔA 340 nm/min. Activity was expressed relative to protein concentration (mg). The anti-venom potency was determined by mixing 5LD50 of B. andianus venom with 12.5, 25, 50, 100 or 200 μl of PABA and incubating for 1 h at 37 °C followed by i.p. injection in 5 groups of 4 mice. Median effective dose (ED50) was calculated from the number of deaths within 24 h of injection of the venom/anti-venom mixture using Probit analysis as described above. The ED50 was expressed as ml anti-venom/mg of venom needed to prevent death in 50% of the injected mice. To determine the neutralization of hemorrhagic activity, PABA was incubated with either acetylcholine 3MHD or 5MHD for 30 min at 37 °C according to manufacturer’s

instructions (1 μL of serum to 2.5 μg of venom) and inoculated in different groups of 3 Swiss male mice (18–22 g) as described above. Positive and negative control groups, each consisting of 2 mice were treated with venom alone (5MHD) or anti-venom, respectively. Two hours later, mice were euthanized and the hemorrhage was measured (Kondo et al., 1960; Sanchez et al., 1992). Inhibition of PLA2 activity of B. andianus venom by PABA was conducted as described by Gutierrez et al. (1998). Two MPD of venom were incubated with 13, 6.5 and 3.25 μl of anti-venom for 30 min at 37 °C, and 15 μl of each mixture added in triplicate to wells in agarose gels. Neutralization of venom was checked by the absence of halos on the plate’s surface. Inhibition of dimethylcasein hydrolysis by PABA was estimated by incubation (30 min at 37 °C) of a fixed concentration of B. andianus venom with increasing amounts of anti-venom (μl). After incubation the mixtures were tested as described before.