Horseradish peroxidase (HRP; EC is the most widely used enzyme label for antibodies. It is relatively small (44 kDa), stable, and has a broad specificity that allows is to be measured by absorption, fluorescence, and luminescence. Several of its products are intensely colored, which makes the enzyme convenient to use for immunocytochemistry and immunoblotting applications.

The most commonly used method (1) for labeling immunoglobin G (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. The conjugates are of a high molecular weight (0.5-1 x 106) because several enzyme molecules can bind to each IgG molecule and also crosslink the latter. Peroxidase has very few free amino groups so self-coupling is not usually a significant problem.

IgG antibody may be labeled with HRP using glutaraldehyde in a two-step procedure (2); this produces small conjugates, but their activity is low. Heterobifunctional reagents such as succinimidyl 4-(N-maleidomethyl)-cyclohexane-l-carboxylate may also be used to link the thiol group of Fab' fragments to an amino group in the enzyme (3).

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