Purification of the antibody from the crude antisera is best carried out by affinity chromatography using the synthetic peptide immunogen as ligand.
If the peptide has an added cysteine residue, it can conveniently be coupled to the chromatography media through the side chain thiol to give the affinity column. Several other methods of coupling the peptide to the column are available for peptides lacking cysteine. After passing the crude antisera through the column, bound anti-peptide antibody can be selectively recovered by eluting with buffers of low and then high pH. Good yields of active antibody are usually obtained by this method.
Alternatively, the whole IgG fraction can be isolated from the crude antisera by affinity chromatography on Protein A or G columns. This can be useful when the antigen is not available for preparing the affinity column.