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.
Background and Aims: In this study we improved the purification of immunoglobulins from equine antiserum raised against the venom of 6 types of scorption species. Caprylic acid (octanoic acid), a fatty acid, was found to have no effect on the activity of the enzymes pepsin, which is used in antivenom purification to digest Fc fragment of immunoglobulins to obtain F(ab´)2. Materials and Methods: A new method was developed for the production of F(ab´)2 antivenom whereby whole equine antiserum was mixed with equal amount of 0.15 M HCl and pH 3.4 with pepsin 660 mg/L of diluted antivenom and incubated for 4 h at 37°C. After digestion the pH were brought to 4.8 with sodium hydroxide solution (1.5 M) and then 1.5% caprylic acid and 10% ammonium sulfate was added and mixed for 60 minutes and passed through filter paper. Results: Caprylic acid caused precipitation of albumin, and ammonium sulfate reduced turbidity of solution, resulting in a reduced protein load presented to the digestion enzymes culminating in substantial reductions in processing time. Conclusions: The equine F(ab´)2 obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step and time consuming conventional salt fractionation with ammonium sulfate.
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