Improved Recovery of Immunoglobulin Fraction from Egg Yolk of Chicken Immunized with AsialoGM1.
Chicken egg yolk is an abundant, yet inexpensive source of polyclonal antibodies. However, removal of the contaminating lipids and lipoproteins from immunoglobulins of egg yolk (IgY) is considered to be a laborious task during the purification. Liposomal asialoGM1 was used as a model immunogen for raising polyclonal IgY and tested with various methodological approaches. To overcome certain difficulties posed during IgY purification, several detergents (cationic, anionic and non-ionic) were tested for their effect on yolk protein and lipoprotein separation during water dilution processing of IgY. Cetrimide showed a pronounced effect on improving the immunoglobulin separation with only less than 3% of the lipid contaminants as compared to other detergents. The least effective detergent was sodium dodecyl sulphate with 14-35% contaminating lipids. The usefulness of egg yolk acetone powder for long-term storage prior to IgY purification was also determined. The use of acetone precipitated proteins for IgY processing showed a 25-30% loss in antigen specific immunoreactivity despite of an optimum total immunoglobulin recovery of 80%. A combination of delipidation of yolk with 50% acetone and treatment of the resultant acetone precipitated protein with a low ionic buffer (pH 5.5) containing 200 &mgr;M of cetrimide yields electrophoretically homogeneous IgY with an optimum total immunoglobulin recovery of 85-90%. The antigen specific immunoreactivity of anti-asialoGM1 IgY was preserved well during the cetrimide treatment and was found to be higher than all other recovery processes, as tested by radial immunodiffusion and immunoblot assays.
Sriram
Venkataraman
V
Medical Biotechnology Research and Development, Research Council-Tamilnad Hospital Academic Trust, Chennai, India.
Yogeeswaran
Ganesa
G