Optimization of purification procedure for horse F(ab´)2 antivenom against androctonus crassicauda (scorpion) venom

Abstract

Purpose: To immunize antiserum of horse with Androctonus crassicauda scorpion venom in order to achieve an antivenom with higher purity by combined caprylic acid/ammonium sulfate. Methods: The optimum pH to terminate enzymatic digestion was evaluated. Purification was performed by various combinations of caprylic acid (0 to 2.5 mL %) and ammonium sulfate (0 to 20 g %) at 25, 30 and 37 ºC. The effects of three factors (caprylic acid, ammonium sulfate and temperature) were evaluated based on precipitation of non-immunoglobulin proteins. Antivenom purity was evaluated by determining the concentration of desired soluble protein and undesired albumin, as well as by turbidity and titration. Results: The results showed that the optimum pH for inhibition of enzyme activity and precipitation of impurities was 4.8. SDS-PAGE revealed that the highest impurity precipitation and lowest protein aggregation was occurred at the combination of 1.5 mL % caprylic acid and 10 g % ammonium sulfate at 37 ºC. Conclusion: The modified method of purification significantly decreases turbidity, albumin impurity concentration and processing time but increased antibody titer and purity of antivenom. Therefore, it is a potentially suitable method for purifying antivenom in commercial production.