Preparation of Avidin Conjugates

Abstract

The high-affinity avidin-biotin system has found applications in different fields of biotechnology, including immunoassays, histochemistry, affinity chromatography, and drug delivery, to name a few. A brief description of avidin and avidin-like molecules, streptavidin, deglycosylated avidin, and NeutraLite avidin is presented in the Chapter 2. With four biotin-binding sites per molecule, the avidin family of proteins is capable of forming tight complexes with one or more biotinylated compounds (1). Typically, the avidin-biotin system is used to prepare signal-amplifying "sandwich" complexes between specificity reagents (e.g., antibodies) and detection reagents (e.g., fluorophores, enzymes, and so on). The specificity and detection reagents are independently conjugated, one with avidin and the other with biotin, or both with biotin, providing synthetic flexibility (2). Avidin conjugates of a wide range of fluorophores, phycobiliproteins, seconday antibodies, microspheres, ferritin, and enzymes commonly used in immunochemistry are available at reasonable prices, making their small-scale preparation impractical and not cost effective (see Note 1). However, conjugations of avidin to specific antibodies, to uncommon enzymes, and to other proteins and peptides are often performed on-site. A general protocol for the conjugation of avidin to enzymes, antibodies, and other proteins is described in this chapter.