Streptavidin is a 60,000 molecular weight with an extraordinarily high affinity for the small molecular weight vitamin, biotin. Because this affinity is over one million times higher than that of antibody for most antigens, the binding of Streptavidin to biotin (unlike antibody-antigen interactions) is essentially irreversible. In addition to this high affinity, the Biotin/ Streptavidin System can be effectively exploited because Streptavidin has four binding sites for biotin and most proteins (including antibodies and enzymes) can be conjugated with several molecules of biotin. These aspects provide the potential for macromolecular complexes to be formed between Streptavidin and biotinylated enzymes.
An immunoperoxidase procedure based on these properties was devised for localizing a variety of histologically significant antigens and other markers. (Hsu SM, Raine L, Fanger H: Am. J. Clin. Pathol. 7 5, 734-738, 1981; Hsu SM, Raine L, Fanger H: J . Histochem. Cytochem. 2 9, 577-580, 1981.) This technique employs unlabeled primary antibody, followed by biotinylated secondary antibody and then a preformed Avidin and Biotinylated horseradish peroxidase macromolecular Complex.
Finetest SABC kit contain Streptavidin and biotinylated horseradish peroxidase reagents, which have been specially prepared to form ideal complexes for immunoperoxidase staining. Although the structure of the Streptavidin : biotinylated horseradish peroxidase complex is still undefined, evidence suggests that it consists of many biotinylated horseradish peroxidase molecules crosslinked by Streptavidin into a three dimensional array. The complex apparently has few exposed biotin residues but retains at least one biotin binding site. Formation of the complex is achieved by mixing Streptavidin and biotinylated horseradish peroxidase in dilute solution and in defined amounts prior to use. After the initial incubation there appears to be little change in the complex as judged by only a marginal increase in immune peroxidase staining sensitivity and the complex remains stable for several hours after formation. The high sensitivity and shorter incubation times reported for the Finetest SABC system are likely due to the number of active horseradish peroxidase molecules associated with the complex and the rapid, irreversible interaction of the complex with biotinylated antibody. The low background staining obtainable with the Finetest SABC Kits is probably due to the high dilutions of primary antisera and other reagents employed in the method, the quality of our affinity-purified biotinylated secondary antibodies, and the specially prepared Streptavidin and biotinylated horseradish peroxidase.