The Avidin-Biotin Complex (ABC) Method and Other Avidin-Biotin Binding Methods

These methods involve, at their core, the vitamin biotin and the protein avidin, which bind together irreversibly. By establishing a biotin link, through avidin, between the horseradish peroxidase enzyme and a secondary antibody reagent, enzyme localization can be achieved at the site of primary antibody interaction with the specimen. These procedures are more universal than the purely immunologic techniques (see Chapter 24), requiring only a biotinylated secondary antibody for each species of primary antibody used. The biotin molecule is small and can be easily conjugated to immunoglobulin by amino substitution at alkaline pH without the loss of immunoglobulin activity. These secondary antibodies are quite inexpensive, readily available commercially, and reactive against immunoglobulin from a wide variety of species. The fact that the binding in this technique is not entirely immunologic and almost irreversible because of the high affinity of avidin for biotin, makes this technique less prone to errors related to assay conditions than other purely immunologic techniques. While the sensitivity provided by these methods is generally better than that obtained with the peroxidase-antiperoxidase (PAP) technique (see Chapter 24), sometimes resulting from fixation or for other reasons, the sample may not be suitable for avidin-biotin methodology (1 ). Tissues may have endogenous biotin or possess biotin-like binding capabilities, which may result in nonspecific binding. The irreversible nature of the binding also allows for the procedure to be undertaken with the most stringent of conditions.