The avidin-biotin complex (ABC) method of IHC staining was developed in 1981. This method is still used to a limited degree in some Pathology laboratories. This method of IHC staining relies upon the strong affinity avidin has for biotin.
Avidin is a large glycoprotein that has a very high affinity for a low molecular weight vitamin called biotin. Avidin has multiple binding sites for biotin and can be chemically conjugated to peroxidase. Biotin is easily conjugated to antibodies and enzymes. Because avidin has an extreme affinity for biotin, the binding of avidin to biotin is essentially irreversible.
The ABC methodology consists of the following steps:
- An unlabeled primary antibody is applied to the tissue section
- A biotinylated secondary antibody is applied, introducing biotin molecules at the site of the primary antibody.
- A complex of avidin and biotin horseradish peroxidase conjugate (ABC) is applied.
- The peroxidase is then developed by the DAB or other chromogen substrates to produce a colored label at the antigen site.
The disadvantage to using the ABC method of IHC staining is the possibility of the avidin-biotin complex binding erroneously with elements that exist naturally within the tissue. Avidin is known to bind with tissue lectins, contributing to non-specific staining. Endogenous biotin artifact is common in the cytoplasm of hepatocytes, as well as in some tumors. Binding of the ABC complex with endogenous biotin could lead to false-positive staining results.
