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

• Published in: Toxicon (Oxford) Vol. 60; no. 6; pp. 1022 - 1029
• Main Authors: Eursakun, Sukanya, Simsiriwong, Pavinee, Ratanabanangkoon, Kavi
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier Ltd 01.11.2012; Elsevier
• Subjects: ammonium sulfate; Ammonium Sulfate - chemistry; Ammonium sulfate-Caprylic acid combination; Animal poisons toxicology. Antivenoms; Animals; antibodies; Antivenins - chemistry; Antivenom; antivenoms; Biological and medical sciences; Caprylates - chemistry; Chemical Fractionation - methods; Chromatography, High Pressure Liquid; Equine antivenom; filtrates; Fractionation; high performance liquid chromatography; Horses; immunoglobulin G; Immunoglobulin G - chemistry; Medical sciences; octanoic acid; titration; Toxicology; turbidity; 3D; Equine antivenom; Fractionation; AS; Antivenom; Ammonium sulfate-Caprylic acid combination; NK3; Vo; NK; CA; Antifungal agent; Association; IgG; Sulfates; Octanoic acid; Ammonium sulfate-Caprylic acid; combination
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2012.07.005

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. ► Antivenom IgG was fractionated by a combination of ammonium sulfate and caprylic acid. ► Ammonium sulfate reduced turbidity and increased the yield but not the purity of IgG. ► Optimum fractionation condition was 3.5% caprylic acid. ► Better overall results were observed with one hour than with overnight precipitation.