REMOVAL OF BOVINE SERUM-ALBUMIN FROM COWS MILK USING CHICKEN EGG-YOLKANTIBODIES 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. Ig Y developed against BSA was reduced by 2-mercaptoethylamine. The react ivities of reduced and whole IgY against BSA were not significantly di fferent. The reduced IgY was covalently linked to chitosan gel through stable covalent thioether linkages using sulfo-succinimidyl-4-(N-male imidomethyl) (sulfo-SMCC) as a cross-linker. The density of antibody I gY immobilized on chitosan gel was approximately 3-5 mg per mi of chit osan gel. The ligand-binding capacity of immobilized IgY towards BSA w as 0.35-0.44 mg BSA per mi of chitosan gel. A single pass of skimmed m ilk through the column allowed the removal of BSA from the milk sample . The milk sample was analyzed, before and after immunoaffinity separa tion, by SDS-PAGE. BSA was desorbed with 0.5 M-glycine-HCl buffer at p H 2.8 but the reusability of the column was limited to three cycles. A lternatively, BSA was desorbed with 0.5 M-glycine-HCl buffer containin g 2 M-NaCl at pH 4.6 after longer incubation times at a slower flow ra te. 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.