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

Abstract

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.