Improvement of purification methods for F(ab')2 fraction of equine hyperimmune plasma against scorpion venom

Antivenoms used for treatment of envenomation caused by like snakes and scorpions, are mostly produced in equine (horses) or ovine (sheep and goat). In Iran, the production of polyspecific scorpion antivenom has been started since 70 y ago and purified with ammonium sulfate fractionation after pepsin digestion of Fc fragment from IgG. Since this conventional method is time consuming, expensive and the product has lower purity. In this study we used either caprilic acid (Octatonic acid) alone or with combination with ammonium sulfate for separation and removal of impurities and precipitation of specific F(ab′)2 against scorpion venom. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed that purification with caprilic acid at a final concentration of 3.5% is optimal to obtain immunoglobulins essentially free of albumin. The second method of purification includes combination of 1.5% (v/v) caprilic acid with 20% saturation with ammonium sulfate. Both procedures are performed after peptic digestion of IgG (removal of Fc fragment) at 37°C for 4 h at pH=3.5. After peptic digestion pH is raised to 4.8 with 1.5 molar sodium hydroxide to stop enzyme digestion then in the first method 3.5-5% caprilic acid was added with strong agitation for one hour the impurities were removed with filter paper and the supernatant was dialyzed against CIS buffer. In the second method after adjusting pH=4.8 ammonium sulfate was added to 20% saturation then 1.5% caprilic acid was added with strong agitation for one hour then slurry is passed through filter paper the precipitated impurities discarded and supernatant was dialyzed