[13] Solid-Phase Assay for d-Biotin and Avidin on Cellulose Disks

This chapter describes the solid-phase assay for d-biotin and avidin on cellulose disks. Most biotin assay procedures exploit the absolute growth requirement of certain bacterial and yeast strains for d-biotin, or depend on the extremely high affinity of avidin for this vitamin. The binding assays, particularly those employing immobilized avidin, are faster and easier to perform. However, when avidin is covalently linked to particulate supports, it becomes difficult to distribute accurately constant amounts of the immobilized protein among different samples and to avoid losing some of the biotin-immobilized-avidin complex while centrifuging or filtering particles to remove unbound biotin. These problems can be overcome by changing the geometry of the avidin-carrying support. The filter paper disks of uniform size, loaded with essentially constant amounts of immobilized avidin, are easily transferable from one aqueous environment to another. Such avidinylated disks of known biotin-binding capacity form the basis of the sequential competition assay in which primary immersion of the disks in a sample of unknown biotin content is followed by exposure to an excess of radioactive biotin. In the event that disks can bind more biotin than test samples contain, decreased disk capacity for the labeled ligand becomes a direct measure of sample biotin content. The source of avidin for preparation of avidin–(biotin–cellulose) disks can be commercial avidin or egg white.