ISOLATION OF LACTOFERRIN BY IMMUNOAFFINITY CHROMATOGRAPHY USING YOLK ANTIBODIES

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Abstract
Immunoaffinity columns for isolation of lactoferrin from milk or cheese whey were prepared by immobilization of egg yolk antibodies. Specific yolk antibodies were obtained by affinity purification of a crude water-soluble fraction or ammonium sulfate-purified immunoglobulin (IgY) from egg yolk of hens immunized against bovine lactoferrin. Specific antibodies against lactoferrin (IgYLf) comprised about 11% of the total IgY. The antibodies were covalently coupled by reductive amination to a commercially beaded agarose with monomeric aldehyde groups, at three different ligand densities (1.5, 3.3 and 9.2 mg per mL resin). In model systems of lactoferrin in phosphate buffered saline, binding capacity expressed as mole percentage of bound lactoferrin to immobilized IgYLf were 34, 32, and 20%, respectively, for the three ligand densities. Binding capacity was increased to 80 ± 25% (n=8) by omitting saline in the equilibrating buffer, and recoveries of 6 ± 2 mg lactoferrin (n=7) were obtained by chromatography of milk sources on a 4.5 mL column containing 14.7 mg IgYLf. SDS-PAGE indicated that lactoferrin could be isolated from cheese whey or raw skim milk without changing other protein components.