Optimization of purification procedure for horse F(ab´)2 antivenom against Androctonus crassicauda (Scorpion) venom
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.
Keywords: Antivenom, Scorpion, Androctonus crassicauda, Purification, Albumin, Antiserum
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