Horseradish Peroxidase Labeling of IgG Antibody

Horseradish peroxidase (HRP; EC 1.11.1.7) is probably the most widely used enzyme label. This protein is relatively small (44 kDa), stable, and has a broad specificity which allows it to be measured by absorption, fluorescence or luminescence. Some of its products are intensely coloured, which makes this enzyme suitable for immunocytochemical or immunoblotting applications. The most popular method (1) for labeling IgG antibody molecules with HRP exploits the glycoprotein nature of the enzyme. The saccharide residues are oxidized with sodium periodate to produce aldehyde groups that can react with the amino groups of the IgG molecule, and the Schiff bases formed are then reduced to give a stable conjugate of high molecular weight (0.5–1.0 × 106). The enzyme has few free amino groups so self-coupling is not a significant problem.