13 - Avidin-Biotin Systems

Abstract

One of the most popular methods of noncovalent conjugation is to make use of the natural, strong binding of avidin to the small molecule biotin. The strength of the avidin–biotin interaction has made it a powerful tool in specific targeting applications and assay design. Since each avidin molecule contains a maximum of four biotin-binding sites, the interaction can be employed to enhance the signal strength in immunoassay systems. Modification reagents that can add a functional biotin group to proteins, nucleic acids, and other molecules now come in many shapes and reactivities. Depending on the functional group present on the biotinylation compound, specific reactive groups on antibodies or other proteins may be modified to create an avidin-binding site. Amines, carboxylates, sulfhydryls, and carbohydrate groups can be specifically targeted for biotinylation through the appropriate choice of biotin derivative. Photoreactive biotinylation reagents are used to nonselectively add a biotin group to molecules containing no convenient functional groups for modification. Similarly, oligonucleotide probes are often modified for detection with avidin or streptavidin conjugates. This chapter discusses the concept and use of the avidin–biotin interaction in bioconjugate techniques. It also reviews the preparation of biotinylated molecules and avidin conjugates and provides the protocols.