Preparation of Peroxidase-Labeled Antibody

Abstract

The first procedure binding antibody to an enzyme was published by Nakane and Pierce (1966). These authors established covalent linkage between the two components by p,p’-difluoro-m,m’-dinitro-diphenyl sulfone. A peroxidase enzyme was used to detect the bound antibody both at light and electron microscopic levels. Nakane and Kawaoi later (1974) developed a new procedure to bind peroxidase to antibody by oxidizing the carbohydrate moiety in the enzyme with periodate, thus forming aldehyde groups which react readily with primary amino groups in the antibodies or Fab’ fragments. The periodate treatment of the enzyme does not interfere with its enzymatic activity and a high percentage of the oxidized enzyme can be bound to antibodies without interfering with the reactivity of the antibody. This procedure is given here, based on the description of Nakane and Kawaoi (1974).