Introduction of Biotin-Avidin-System
Biotin-Avidin-System (BAS) developed in the late 70s, is a new type of bioreaction amplification system. It can bind with almost any marker that has been developed successfully. With the firm bond between Biotin-Avidin and marker, as well as the multistage amplification effect, BAS makes immune labelling technic and related tracer analysis more sensitive. BAS is now widely applied in qualitative or quantitative detection and localization observation of antigen or antibody.
Biotin (B), widely distributed in animal and plant tissues, is often extracted from egg yolk and liver tissue rich in biotin. Biotin has a molecular weight of 244.31Da with two ring structures (Figure1): imidazolone ring (I), the main site binding with avidin, and thiophene ring (II) with the pentanoic acid as the side chain at C2, whose carboxyl terminal is the only structure to bond with antibody and other biomacromolecules. After chemical modification, the biotin becomes a derivative with various active groups, namely activated biotin (Figure 2).
Avidin (AV), also known as antibiotin, is a type of alkaline glycoprotein extracted from ovalbumin. With a molecular weight of 68kD, each avidin molecule consists of 4 identical subunits, therefore binding with 4 biotin molecules at most. Avidin has strong affinity for biotin, with a binding constant (K) up to 1015 L/mol, 10,000 times more than that between antigen and antibody (K=10.5~11 L/mol). As a result, their bond is very stable with high specificity.
The great advantages of BAS in practical application are as follows.
Biotin is prone to bond with protein, nucleic acid and other biomacromolecules. The derivative it forms not only maintains the original bioactivity of the biomacromolecule, but also exhibits multivalence. Besides, each avidin molecule has 4 binding sites for biotin, so it can bond with biotinylated macromolecule derivative and marker at the same time. Therefore, the multistage amplification effect of BAS makes it highly sensitive in practical application.
The avidin has strong affinity for biotin with high specificity. Therefore, the multistage amplification effect of BAS improves the sensitivity without adding non-specific interference. The binding properties of BAS won’t be affected by highly dilution. As a result, the non-specific effect of reaction reagents can be minimized.
The Biotin-Avidin binding constant is a million times more than that of antigen-antibody reaction. The Biotin-Avidin compound has a low dissociation constant. This process is irreversible and free from the effect of acid, alkali, denaturant, proteolytic enzyme and organic solvent. Therefore, the product of BAS in practical application is stable, reducing the operation error thus improving the precision of detection.
Biotin and avidin can both form various derivatives. Not only can these derivatives be used to detect antigen-antibody, hormone-receptor, nucleic acid system in body fluid, tissue and cell and other bioreaction system by combining with different labeling techniques based on enzyme, fluorescein and radionuclide, but also they can be made into affinity medium for separation and purification of the reagents in above system. Besides, diversified commercial reagents made from BAS provide favorable conditions for clinical tests and scientific research.
BAS can produce various universal reagents such as biotinylated secondary antibodies according to specific experiment requirement, which can be applied to different reaction systems. They can also be highly diluted before use with less amount and low cost. Particularly, when BAS combines with expensive antigen-specific primary antibody, it can greatly reduce the latter dosage and therefore save the cost. Besides, biotin bonds with avidin very quickly and efficiently. Despite the multilevel reactions, it takes only several hours for the whole experiment with short time of incubation.
Biotin labeling kit