Removal of bovine serum albumin from cow's milk using chicken egg-yolk antibodies immobilized on chitosan gel

Polyclonal chicken antibodies raised against bovine serum albumin (BSA) were immobilized on chitosan gel for the immunoaffinity isolation of BSA from cow's milk. Antibodies (IgY) against BSA were isolated from egg-yolk, purified and antibody reactivity to antigen was measured. IgY developed against BSA was reduced by 2-mercaptoethylamine. The reactivities of reduced and whole IgY against BSA were not significantly different. The reduced IgY was covalently linked to chitosan gel through stable covalent thioether linkages using sulfo-succinimidyl-4-(N-maleimidomethyl) (sulfo-SMCC) as a cross-linker. The density of antibody IgY immobilized on chitosan gel was approximately 3-5 mg per mi of chitosan gel. The ligand-binding capacity of immobilized IgY towards BSA was 0.35-0.44 mg BSA per mi of chitosan gel. A single pass of skimmed milk through the column allowed the removal of BSA from the milk sample. The milk sample was analyzed, before and after immunoaffinity separation, by SDS-PAGE. BSA was desorbed with 0.5 M-glycine-HCl buffer at pH 2.8 but the reusability of the column was limited to three cycles. Alternatively, BSA was desorbed with 0.5 M-glycine-HCl buffer containing 2 M-NaCl at pH 4.6 after longer incubation times at a slower flow rate. The low ligand-binding capacity was not an impedement to reuse of the column. The column was reused more than 20 times with minimal loss of binding capacity.