Studies on the fractionation of equine antivenom IgG by combinations of ammonium sulfate and caprylic acid

Abstract

This study involved the use of combined stepwise ammonium sulfate (AS) and caprylic acid (CA) fractionation of equine antivenom IgG without intermediate separation of precipitate. Using a microplate and checker board titration format, plasma was treated under 66 conditions with varying concentrations of AS (0–25% saturation) and CA (0–5% v/v). The filtrate of each well was assayed for protein and antibody activity. At about 1.5–4.0% CA without AS, the precipitated plasma gave high specific antibody activity.Twelve precipitation conditions selected from the microplate experiment were studied in detail in tubes. The highest turbidity was with 5% CA alone. The highest antibody recovery of 95.45% was observed at 15% AS with 3.0% CA. The highest specific activity with 3.28 folds purification was observed with 4.0% CA. Thus, AS could reduce the turbidity induced by CA and increase the yield but not the purity of antibody. Size exclusion HPLC showed the antibody to be one single peak with 1.5% of soluble protein aggregate.When all parameters were considered, the optimum fractionation condition appeared to be 3.5% CA alone which gave high specific antibody activity (3.26 folds purification), antibody recovery (93.93%) and low turbidity (0.56% solid). Furthermore, better overall results were observed with one hour than overnight precipitation.