Bothrops alternatus venom pretrated with chelate agent for antiserum production

Abstract

Snakebite envenoming is a major public health problem in tropical countries. The only specific therapies currently available for the treatment are antivenoms, which consist of polyclonal immunoglobulins purified from sera/plasma of horses or sheep immunized with doses sublethal of snake venom(s). Bothrops alternatus (BaV) venom induces a prominent tissue local damage, especially, hemorrhage, muscle damage and inflammation. These complex pathological phenomena are due to the concomitant action of metalloproteinases (SVMPs) in venoms and others edema-inducing components. In order to obtain a high titer of antibodies and reduced local damage in the animal, an alternative immunization protocol was proposed where the SVMPs action was blocked by using a chelate agent. For this proposal, the B. alternatus venom (10 mg/mL) was blocked by EDTA-Na2 (10 mM, BaV/ EDTA-Na2) and used as antigen. Previously to the inoculation, the excess of chelate was removed by passing the mixture on Sephadex G-25 column (venom without inhibitor was subjected to the same process) and the effective neutralization of SVMPs using azocasein as substrate was determined. Group of 5 Balb/c mice were immunized subcutaneously on days 0, 15 and 30 with BaV(15-30-45μg) or BaV/ EDTA-Na2 (45-90-135 μg) emulsified with Freund?s Adyuvant (complete first and incomplete-booster). Blood samples were collected by the animals tail tip on days 14, 29 and 41 of protocol immunization and it was destined to ELISA´s test. The results showed that the immunized animal with BaV/ EDTA-Na2had a higher titer (5.1x104) than those treated to BaV (1.3x104). Macroscopic analysis at the inoculation site of mice injected with Freund?s adjuvant showed local damage (with non-infectious abscesses) and hypertrophy of inguinal lymph nodes. Our results show that BaV/ EDTA-Na2 formulation, where the SVMPs are blocked, produced a higher humoral response compared with the produced by BaV. These preliminary results demonstrated the potential use of blocking the toxins with chelate to produce antivenom with less damage in the animals.