Preparation of Monomeric Fab'‐Horseradish Peroxidase Conjugate Using Thiol Groups in the Hinge and Its Evaluation in Enzyme Immunoassay and Immunohistochemical Staining. a

Horseradish peroxidase and Fab' were conjugated by using thiol groups in the hinge of Fab'. Maleimide or pyridyl disulfide groups were introduced into peroxidase by treatment with N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or N-succinimidyl 3-(2-pyridyldithio)propionate and were allowed to react with thiol groups in the hinge of Fab'. The conjugates were obtained in high yields with a minimal polymerization and without impairing the activities of peroxidase and antibodies, and were superior to those prepared using amino groups of Fab' by the glutaraldehyde and periodate methods in performing sandwich enzyme immunoassay and immunohistochemical staining. The conjugate yield was higher in the maleimide method than in the pyridyl disulfide method.