Potential low-impact immunogen for the production of anti-bothropic serum: Bothrops alternatus venom treated with Na2EDTA.

This study proposes an alternative method using Na2EDTA to neutralize B. alternatus venom and using it as an immunogen from the start of inoculation to minimize side effects and enhance antivenom production. To achieve this, 1.8 mg/mL of B. alternatus venom (B.aV) was treated with Na2EDTA, and any extra chelate was eliminated by filtering the resulting solution through a Sephadex G-25 column. Two groups of BALB/c mice were immunized subcutaneously on days 1, 15 and 30 with B.aV/Na2EDTA (45, 90, 135 µg/mouse) or B.aV (15, 30, 45 µg/mouse), respectively. Both formulations were emulsified with Freund's adjuvant (complete first and incomplete-booster). Blood samples were collected from each mouse on days 14, 29, 41, and 50 post-first immunization, and serum was separated for antibody detection. Animals were then sacrificed and lungs removed for histological analysis (hematoxylin-eosin). Immunoblotting analysis revealed that the sera from mice inoculated with B.aV/Na2EDTA (anti-B.aV/Na2EDTA) recognized the major venom proteins (20-66 kDa) similarly to the sera from mice inoculated with B.aV (anti-B.aV). The enzyme-linked immunosorbent assay results indicated that the anti-B.aV/Na2EDTA had a higher titer (5.76 × 104) than those the anti-B.aV (1.92 × 104). Additionally, sera from animals immunized with B.aV/Na2EDTA significantly neutralized proteolytic, indirect hemolytic and coagulant activity (p < 0.05). Finally, histological examination of the lungs of mice inoculated with B.aV/Na2EDTA showed normal appearance, while animals inoculated with B.aV showed interstitial lung injury (p < 0.05). In conclusion, the B.aV/Na2EDTA formulation, free of excess Na2EDTA, proved to be a promising candidate as an immunogen for antivenom production.

New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2.

Antivenoms are usually obtained by animal immunization with successive inoculations of increasing sublethal amounts of venom, which may impair the animal health. The high lethality of venom requires prolonged immunization plans with small amounts of venom. Thus, we propose an alternative plan that includes a pre-immunization of the animal with phospholipase A2, the main crotoxin component, which is responsible for the whole venom lethality. For comparison, three different immunization schemes were designed: high dose protocol (HDP; 0.5-27 mg of venom), low dose protocol (LDP; 0.1-7 mg of venom) and Mix protocol (MP; preimmunization 0.1-1.2 mg of crotalic PLA2, and then 4.5-8 mg of venom). Antibody titers were determined by ELISA, in blood plasma obtained from the marginal vein of the ear. The neutralizing ability of the different sera obtained by all protocols (HDS, LDS and MS) was tested against the most important pharmacological activities of whole venom: PLA2 activity, myotoxicity, thrombin like activity and lethality. MS showed the best neutralizing efficacy and at the same time, it was obtained by an immunization protocol that takes account of animal health care, since it requires low quantities of venoms in comparison to traditional protocols.