Perioxidatic Reaction of Catalase-Antibody Complex of Leukocytes Demonstrated by Diaminobenzidine¹

A new immunochemical procedure using the chromogenic substrate, 3,3′-diaminobenzidine tetrahydrochloride (3,3′-DAB), was developed to elucidate the immunological interrelationships of peroxidatic catalases in normal peripheral blood leukocytes, in leukocytes of a patient with myelogenous leukemia, and in normal erythrocytes. Catalase of normal leukocytes was shown to have only one weakly enzymically active component disclosed by the 3,3′-DAB reaction in Ouchterlony double diffusion tests. Normal erythrocytes and peripheral blood leukocytes of a patient with myelogenous leukemia, in contrast, revealed two components: a weak inner component and an intensively reactive outer component. The inner components of the latter cells showed a reaction of immunological identity with themselves and with the component of normal leukocytic catalase. The outer, intensely 3,3′-DAB-positive reaction of normal erythrocytes and leukemic leukocytes showed complete immunological identity.

These observations disclose that peroxidatic catalase may be specifically identified when immunological tests are conducted on agarose gel in the presence of 3,3′-DAB. The latter acts as a hydrogen donor and forms an insoluble precipitate by the peroxidatic action of catalase reacting on the substrate, H₂O₂. The data also suggest that catalase in peripheral leukocytes is modified in myelogenous leukemia and becomes identical to erythrocytic catalase, thus losing some of the physical characteristics of normal leukocytic catalase.