The SA/rABC technique: A new ABC procedure for detection of antigens at increased sensitivity

Abstract

Since its introduction, the avidin-biotin-peroxidase (ABC) complex has become an invaluable detection system for a wide variety of bioanalytical applications. In these techniques, avidin and biotin peroxidase are mixed at a pre-determined ratio so that the soluble ABC complex retains biotin binding sites. Consequently, the complex contains an excess of avidin over biotinylated peroxidase residues. On theoretical considerations, however, an ABC complex designed for maximal signal intensity must consist of an excess of peroxidase over avidin molecules. Therefore, ABC complexes with reversed molar ratios of biotinylated peroxidase to avidin (rABC complexes) were prepared and an intermediate streptavidin step was introduced to bind the rABC complexes to biotinylated IgG molecules. The signal generating power of this new streptavidin-rABC sequence proved superior to that of the conventional ABC technique in ELISA assays and in immunostaining of tissue sections.