Antibodies bind specifically to the antigens that induce their production. This specificity is an advantage when identifying and quantifying proteins in an unknown sample mixture. This is the basis for immunoassays such Western blots and enzyme linked immunosorbent assays (ELISA). However, in some cases structural similarities between proteins and interference from diluent ingredients can lead to nonspecific binding resulting in high background and false positive signals. This is very common problem with polyclonal antibodies, and in multiplexed assays.
Biotin and streptavidin offer solution for improving specificity and reducing background without disrupting the normal biological function of the proteins. Biotin (Vitamin B7) is a water-soluble vitamin which covalently binds to and stabilizes proteins in a biological system. Due to its small molecular weight and simple chemical structure biotin conjugation does not affect the biological function of bound proteins.
Biotin also has high affinity for the tetrameric proteins avidin and streptavidin which is structurally designed to bind to biotin via hydrogen bonding. Although non-covalent, the biotin-streptavidin bond is stable under normally denaturing pH and temperature conditions.
The biochemical affinity between biotin and streptavidin is used to create an elegant protein linking system to resolve non-specific binding in immunoassays. Biotinylation (i.e. biotin labeling) of a secondary antibody in sandwich ELISA followed by use of streptavidin conjugated detection reagents such as Streptavidin-HRP (Horseradish peroxidase enzyme) boosts specificity and reduces background signal of the assay.
Efficient biotin labeling can be achieved using primary amine and sulfhydryl groups of target proteins. Conjugation of biotinylation agents to amino acids that regulate protein activity should be avoided. Proteins usually have multiple reactive sites from which to choose. Potential targets for effective biotinylation are N-terminus primary amines (-NH2) , C-terminus carboxyls (-COOH) and glycoprotein carbonyls (-CHO) groups.