Peroxidase-labeled antibody. A new method of conjugation

TLDR: A new method of conjugating horseradish peroxidase with proteins was developed by oxidizing the carbohydrate moiety with sodium periodate and bound to free amino groups of proteins unidirectionally at high efficiencies.

Abstract: A new method of conjugating horseradish peroxidase with proteins was developed. The carbohydrate moiety of fluorodinitrobenzene-blocked peroxidase was oxidized with so- dium periodate to form aldehyde groups. The peroxidase-aldehyde was then bound to free amino groups of proteins unidirectionally at high efficiencies. Peroxidase-labeled immune- globulin retained its immunologic as well as enzymatic activities. Horseradish peroxidase (HRPO), when cou- pled to immunoglobulin G (IgG), has proven to be a useful marker for immunohistochemistry (13). In contrast to other immunohistochemical markers, such as fluorescein (7) and ferritin (20), HRPO may be used for both light and electron microscopy. It is especially suitable for the intracellular localization of antigens at the ultrastructural level since HRPO (40,000 molec- ular weight) is considerably smaller than fer- ritin (650,000 molecular weight); thus, HRPO- labeled IgG (HRPO-IgG) has superior penetra- tion properties. HRPO is usually coupled to IgG using bifunc- tional reagents. With earlier methods, the con- jugation reactions were carried out in the pres- ence of HRPO, IgG, and p,p'-difluoro-m,m - din tro-diphenyl sulfone (FNPS) (13), (1-cyclohexyl- 3-(2-morpholinoethyl)) carbodiimide metho-p- toluenesulfonate (4), cyanuric chloride (1), bis-