The Avidin-Biotin Complex(ABC) Method

This method, described in 1981 by Hsu and associates, makes fundamental use of the covalent and irreversible binding seen between avidin, an egg white protein, and biotin, a vitamin (1 ). 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 (see ) Fig. 1 ). The biotin molecule is small and can be easily conjugated to immunoglobulin by amino substitution at alkaline pH without the loss of immunoglobulin activity. The horseradish peroxidase enzyme can incorporate many biotin molecules without the loss of enzymatic activity. Biotin can also be conjugated to immunoglobulin, which accepts the molecules, as did the enzyme, without the loss of apparent activity (2 ). The two biotin molecules can be joined via an avidin molecule by creating a complex of avidin and biotinylated enzyme, and attaching it to the biotinylated secondary antibody. There are four binding sites for biotin on each avidin molecule and two binding sites for avidin on each biotin molecule. Together, the biotinylated enzyme and avidin molecules form a lattice complex and remain in solution. The ratio is such that there is always an available biotin-binding site on the avidin-biotin complex for binding of the biotinylated secondary antibody.