Colloidal Gold/Streptavidin Methods

Biotin-avidin detection systems are widely used in both immunocytochemistry and molecular biology (1 , 2 ) (see Chapter 21 ). They take advantage of the high affinity of biotin, a low-molecular-weight vitamin, for avidin, an egg-white protein. The avidin-biotin complex has one of the highest dissociation constants known, 10−15 M . This high dissociation constant has made it a convenient system for linking indicators to antibodies. However, egg-white avidin binds nonspecifically to many tissue sites. This nonspecific binding has been attributed both to its high isoelectric point (PI = 10) and to the fact that the protein is glycosylated (3 , 4 ). Because of the problem of nonspecific binding of avidin, streptavidin has largely replaced avidin for immunocytochemical procedures. Streptavidin, produced by Streptomyces avidinni , has properties that are very similar to avidin, but it is not glycosylated (5 ). Streptavidin generally gives little or no background staining, and is superior to avidin for immunocytochemical uses.