Inhibition of mutalysin II, a metalloproteinase from bushmaster snake venom by human alpha2-macroglobulin and rabbit immunoglobulin.

Mutalysin II is a 22.5-kDa zinc endopeptidase isolated from Lachesis muta muta snake venom. In order to determine whether the inhibitors human alpha2-macroglobulin (alpha2-M) and rabbit antibody to mutalysin II share a common mechanism, we have investigated the inhibition of mutalysin II by these two different glycoproteins. The proteolytic activity of mutalysin II with dimethylcasein as substrate was completely inhibited by human alpha2-M and by a purified rabbit antibody to mutalysin II. The protection of fibrin(ogen) digestion by alpha2-M was slightly better than the protection offered by the antibody. In addition, the purified antibody reacted only with the metalloproteinase in bushmaster venom, as demonstrated by immunodiffusion. SDS-PAGE analysis of reduced samples showed that the interaction of mutalysin II with alpha2-M resulted in the formation of high molecular complex ( approximately 180000) and M(r) 90000 fragments generated by the venom enzyme. Also, fragments at 85 and 23 kDa were detected under non-reducing conditions after incubation of rabbit immunoglobulin with enzyme. Proteolysis of dimethylcasein as substrate revealed that the stoichiometry of inhibition was 1.0 mol of human alpha2-M and 1.5 mol of rabbit IgG antimutalysin II per mole of enzyme. Furthermore, dimethylcasein hydrolysis indicated that several viperid snake venoms, including Bothrops atrox, B. alternatus and Trimeresurus flavoviridis cross-reacted with the specific rabbit antibody to varying degrees.

Determination of the neutralizing potency of horse antivenom against bothropic and crotalic venoms by indirect enzyme immunoassay.

The use of ELISA to determine antisnake venom potency of horse immune sera should provide benefits of costs and reproducibility compared to in vivo assays. In the present investigation we evaluated the correlation between ELISA antibody levels and in vivo neutralization assays. For the indirect ELISA method, 0.016 micrograms/well of Bothrops jararaca or Crotalus durissus terrificus venom were used to coat the plates and 100 microliters/well of each sample of antibothropic or anticrotalic venom sera were used at 1:10,000 dilution. Sheep anti-horse IgG conjugated to peroxidase was added and the substrate H2O2/o-phenylenediamine produced the color that was read at 492 nm. A correlation coefficient of r = 0.97 was found for anticrotalic venom antibodies and no significant correlation was observed for antibothropic venom sera using 16 serum samples from immunized horses. However, when three antibothropic venom sera showing high in vivo neutralization potency and low absorbance in ELISA or high absorbance values and low in vivo protection were not included in the correlation analysis the coefficient value was r = 0.88. The correlation coefficient did not improve for all 16 antibothropic sera when a partially purified Bothrops jararaca venom fraction was used to coat the ELISA plates. The results indicate that ELISA could be used to determine the neutralizing potency of anticrotalic venom sera. For the antibothropic venom sera further studies are needed.

Determination of the neutralizing potency of horse antivenom against bothropic and crotalic venoms by indirect enzyme immunoassay

The use of ELISA to determine antisnake venom potency of horse immune sera should provide benefits of cost and reproducibility compared to in vivo assays. In the present investigation we evaluated the correlation between ELISA antibody levels and in vivo neutralization assays. For the indirect ELISA method, 0.016 µg/well of Bothrops jararaca or Crotalus durissus terrificus venom were used to coat the plates and 100 µl/well of each sample of antibothropic or anticrotalic venom sera were used at 1:10,000 dilution. Sheep anti-horse IgG conjugated to peroxidase was added and the substrate H202/o-phenylenediamine produced the color that was read at 492 nm. A correlation coefficient of r=0.97 was found for anticrotalic venom antibodies and no significant correlation was observed for antibothropic venom sera using 16 serum samples from immunized horses. However, when three antibothropic venom sera showing high in vivo neutralization...