title: 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.
authors: Chulabhorn Graduate Institute, Thailand.
authors: Sukanya
authors: S
authors: Eursakun
authors: Pavinee
authors: P
authors: Simsiriwong
authors: Kavi
authors: K
authors: Ratanabanangkoon
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry*; D000818:Animals; D000997:Antivenins / Q000737:chemistry*; D002210:Caprylates / Q000737:chemistry*; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D006736:Horses; D007074:Immunoglobulin G / Q000737:chemistry*
chemical_list: D000997:Antivenins; D002210:Caprylates; D007074:Immunoglobulin G; C031492:octanoic acid; D000645:Ammonium Sulfate