Use of venom blocked with chelating agent for anti-bothropic serum production

Abstract

Snakes belonging to genus Bothrops are responsible for more than 85% of the bites occurring in South America. Bothrops alternatus is a pitviper widespread in this area and it is one of the most important species associated to snakebites not only in Argentina but also Brazil. Antivenoms are the only specific treatment for envenoming by snakebites. They are made by animals (e.g. horses or sheep) immunizations with sublethal doses of venom. Snake Venom Metalloproteinases (SVMPs) play an important role in envenomation, causing relevant local effects such as hemorrhage, edema and myotoxicity as well as systemic bleeding. They represent around ̴43.1% of the protein composition from B. alternatus venom and causing lesions during the immunization of animals. In this work, an alternative immunization protocol was developed in mice where SVMPs activity was previously blocked by Na2EDTA as chelating agent. For this proposal, the B. alternatus venom (BaV, 10 mg/mL) was treated with 50 mM Na2EDTA (1 h, 37°C) and excess chelator was removed by Sephadex G-25 chromatography. Proteolytic activity was assayed to control the block processing. Groups of 5 BALB/c mice were immunized s.v. on 0-15-30 days with BaV (15-30-45μg) and BaV/Na2EDTA (45-90-135 μg). Blood samples were collected on days 14-29-41 for antibody analysis. Sera from BaV/Na2EDTA protocol have a titer (5.1x104) higher than those treated with BaV (1.3x104). The neutralizing ability of both antivenoms was tested against proteolytic, coagulant and PLA2 activity, resulting that it was significantly higher (p <0.05) for sera from mice bowing to blocked venom. Histological analysis of mice lungs showed that pulmonary parenchyma from BaV immunized mice was significantly affected in relation to those BaV/Na2EDTA treated. In conclusion, BaV/Na2EDTA showed to be an appropriate immunogen, not only proved that serum has a high neutralizing capacity but also has a lower organic impact in animals to antivenom production.