Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) are the twoenzymes most commonly used for protein detection. Enzyme-conjugates offer the most flexibility in detection as an array of chromogenic, fluorogenic, and chemiluminescent substrates are available for use with either enzyme. Of the three, chromogenic substrates are the simplest to use, cost-effective, and provide direct visualization of signal development (i.e., enzyme-substrate reaction results in colored precipitate). Chemiluminescent substrates differ from other substrates in that the enzyme-substrate reaction produces light as a byproduct. In well-optimized assays, chemiluminescent reactions can produce a stable output of light for several hours that can be detected using X-ray film or digital imaging equipment, such as a charged-coupled device (CCD). Compared to the other enzymatic methods, chemiluminescence detection is the most widely used and offers the greatest sensitivity, providing pictogram to femtogram-level detection.

When a higher degree of signal amplification and detection sensitivity is needed, poly-HRP secondary antibodies can be used instead of traditional HRP conjugates. The HRP heteropolymer core conjugated to secondary antibodies drastically increases the molar ratio on the secondary antibody without affecting its functionality. This allows more HRP molecules to be available at the immune complex to react with the substrates developing more desirable signals.