Protein Protocols Protein Protocols

Preparation of Avidin Conjugates 365
49
Preparation of Avidin Conjugates
Rosaria P. Haugland and Mahesh K. Bhalgat
1. Introduction
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 NeutrAvidin avidin, is presented in Chapter
48. 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, secondary
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 onsite.
A general protocol for the conjugation of avidin to enzymes, antibodies, and other
proteins is described in this chapter.
Avidin conjugates of oligodeoxynucleotides are hybrid molecules that not only provide
multiple biotin binding sites, but can also be targeted to complementary DNA or
RNA sequences, by annealing interactions. Such conjugates are useful for the construction
of macromolecular assemblies with a wide variety of constituents (3). The
protocol outlined in Subheading 3.1. can be modified (see Note 2) for the conjugation
of oligonucleotides to avidin.
Streptavidin conjugates are also being evaluated for use in drug delivery systems. A
two-step imaging and treatment protocol has been developed that involves injection of
a suitably prepared tumor-specific monoclonal antibody, followed by a second reagent
that carries an imaging or therapeutic agent, capable of binding to the tumor-targeted
antibody (4). Owing to complications associated with the injection of radiolabeled
biotin (5), conjugation of the imaging or therapeutic agent to streptavidin is being con-
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
365