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

This study proposes an alternative method using Na(2)EDTA 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 Na(2)EDTA, 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/Na(2)EDTA (45, 90, 135 mu g/mouse) or B.aV (15, 30, 45 mu g/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/Na(2)EDTA (anti-B.aV/Na(2)EDTA) 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/Na(2)EDTA had a higher titer (5.76 x 10(4)) than those the anti-B.aV (1.92 x 10(4)). Additionally, sera from animals immunized with B.aV/Na(2)EDTA significantly neutralized proteolytic, indirect hemolytic and coagulant activity (p < 0.05). Finally, histological examination of the lungs of mice inoculated with B.aV/Na(2)EDTA showed normal appearance, while animals inoculated with B.aV showed interstitial lung injury (p < 0.05). In conclusion, the B.aV/Na(2)EDTA formulation, free of excess Na(2)EDTA, proved to be a promising candidate as an immunogen for antivenom production.