Figure 1.
SDS-polyacrylamide gel eletrophoresis.
Samples (20 µg) of M. ibiboboca, M. lemniscatus, M. fulvius, M. altirostris, M. spixii, M. surinamensis, M. corallinus, M. frontalis and M. hemprichii venoms were analyzed by SDS-PAGE in a gradient gel (7.5% to 15%) and silver stained.
Table 1.
Lethal dose 50% (LD50) of Micrurus spp snake venoms determined in murine model.
Figure 2.
Determination of the phospholipase A2 activity.
Samples of individual Micrurus venoms (4 µg), or a mixture of M. frontalis (2 µg) and M. corallinus (2 µg), venoms were incubated for 20 min at 37°C with 180 µL of a mixture containing 5 mM Triton X-100, 5 mM phosphatidylcholine, 2 mM HEPES, 10 mM calcium chloride and 0.124% (wt./vol) bromothymol blue dye in water. Results are representative for three separate experiments and expressed as nanomoles acid per minute per µg of venom.
Figure 3.
Determination of the proteolytic activity.
Samples of Micrurus venoms (50 µg), or a mixture of M. frontalis (25 µg) and M. corallinus (25 µg), were incubated at 37°C with the FRET substrate, Abz-FEPFRQ-EDnp, and the hydrolysis measured in a spectrofluorimeter. Results are representative for three separate experiments and expressed as units of free fluorescence per minute per µg of venom (UF/min/µg).
Figure 4.
Determination of the hyaluronidase activity.
Samples of Micrurus venoms (30 µg), or a mixture of M. frontalis (15 µg) and M. corallinus (15 µg), were incubated for 15 min at 37°C with the hyaluronic acid as substrate. After this period, it was added cetyltrimethylammonium bromide, to develop the turbidity in the mixtures, and the absorbance measured in a spectrophotometer at λem 405 nm. Results are representative for three separate experiments and expressed in units of turbidity reduction (UTR) per mg of venom.
Figure 5.
Cross-reactivity of coral snake antivenom.
[A] ELISA: plates were coated with 10 µg of Micrurus venoms and incubated with different dilutions of coral snake antivenom, followed by GAH/IgG-HRPO, diluted 1∶3,000. The absorbance of the samples was determined at 492 nm. The data presented correspond to the mean OD492 value +/− SD of experiments carried out in duplicate. [B] Western blot: Samples (20 µg) of M. ibiboboca, M. lemniscatus, M. fulvius, M. altirostris, M. spixii, M. surinamensis, M. corallinus, M. frontalis and M. hemprichii venoms were separated by SDS-PAGE in gradient gel (7.5% to 15%), electrotransfered to a nitrocellulose membrane and incubated with the coral snake antivenom diluted 1∶2,000 followed by GAH/IgG-AP. The reaction was revealed with NBT and BCIP.
Figure 6.
In vitro serum neutralization assays.
[A] Phospholipase A2 activity: Samples of the venoms (4 µg) were incubated with the coral snake antivenom (1∶10) for 20 min at room temperature. As positive control, the mixture of M. corallinus (2 µg) and M. frontalis (2 µg) venoms, used for the production of the antivenom, was used. [B] Proteolytic activity: Samples of Micrurus venoms (50 µg) and the coral snake antivenom (1∶4) were incubated for 10 min at room temperature. As positive control, the mixture of the M. corallinus (25 µg) and M. frontalis (25 µg) venoms was used. [C] Hyaluronidase activity: Samples of Micrurus venoms (30 µg) and the coral snake antivenom (1∶20) were incubated for 15 min at room temperature. As positive control, the mixture of the M. corallinus (15 µg) and M. frontalis (15 µg) venoms was used. Venoms residual toxic activities were measured as described in materials and methods. Results are representative for three separate experiments and expressed as percentage of neutralization of the venoms enzymatic activities.
Figure 7.
Antivenom neutralization of Micrurus venoms lethal toxicity.
Samples corresponding to 2 LD50, of each Micrurus venom, were mixed with serial dilutions of the coral snake horse antivenom. The mixtures were incubated for 30 min at 37°C and the animals were i.p. inoculated. The effective dose (ED50) was calculated, from the number of deaths within 48 h of injection of the venom/antivenom mixture, using probit analysis and expressed as mL of antivenom per µg of venom.
Table 2.
Summary of the biological activities of Micrurus spp venoms, cross-reactivity and neutralization potential of the Brazilian coral snake antivenom.