Abstract Anti-peptide antibodies against the C-terminal region of α -amylase were originated by immunizing two kinds of peptide antigen and were used to discriminate between two isozymes of α -amylases. The anti-peptide antibodies only reacted with α -amylase that had the C-terminal region corresponding to the peptide antigens, and could purify the target α -amylase from the mixture of the isozymes. The adsorption and elution behaviors of the anti-peptide antibodies differed depending on the characteristics of the peptide antigen used for immunization. Peptide immunization can provide suitable affinity ligands for the purification and detection of target proteins from contaminants that exhibit high homology with the targets.