Biotin-Avidin-System (BAS) is a new biological reaction amplification system developed in the late 1970s. With the advent of various biotin derivatives, BAS was soon widely used in various fields of medicine. In recent years, a large number of studies have confirmed that the biotin-avidin system can be combined with almost all kinds of markers that have been successfully studied. The strong binding of biotin and avidin with high affinity and multi-stage amplification effect make BAS immunolabeling and related tracer analysis more sensitive. It has become a new technology widely used in the qualitative and quantitative detection of trace antigens and antibodies, as well as in localization observation research.
Principle
Biotin (biotin, B) is widely distributed in animal and plant tissues, and is often extracted from egg yolk and liver tissues with a high content, with a molecular weight of 244.31 Da. The biotin molecule has two cyclic structures (as shown in Figure 1), of which the I ring is an imidazolone ring, which is the main site for binding to avidin; the II ring is a thiophene ring, with a valeric acid side chain on C2, and its terminal carboxyl group It is the only structure that binds antibodies and other biological macromolecules. After chemical modification, biotin can become a derivative with various active groups – activated biotin. Activated biotin can be coupled with almost all known biological macromolecules, including proteins, nucleic acids, polysaccharides, lipids, etc., under the mediation of protein cross-linking agents.
Avidin (AV), also known as avidin and avidin, is a basic glycoprotein composed of 4 identical subunits extracted from ovalbumin, with a molecular weight of 68kD and an isoelectric point pI=10.5 ; Heat-resistant and resistant to the action of a variety of proteolytic enzymes. Especially when combined with biotin, the stability is better. The interaction between biotin and avidin is the strongest known non-covalent interaction. The affinity constant (K) is 1015mol/L, which is at least 1 higher than the affinity between antigen and antibody (K=105～1011mol/L). million times. In addition, the combination of the two has good stability and specificity, and is not affected by reagent concentration, pH environment, or organic solvents such as protein denaturants. Since each avidin can bind 4 molecules of biotin, this feature can be used to construct a multi-level signal amplification system. Therefore, BAS can be used for quantitative, qualitative detection and localization observation of trace antigens, antibodies and receptors, and can also be made into affinity media for the separation and purification of reactants in the above-mentioned various reaction systems.
Since avidin has a sugar chain side chain, it is easy to have non-specific affinity with polysaccharides on the cell surface. Therefore, streptavidin was developed.
Streptavidin (streptavidin, SA) is a protein secreted by Streptomyces avidinii with a molecular weight of 65kD. The streptavidin molecule consists of 4 identical peptide chains, each of which can bind a biotin and does not carry any sugar group, so like avidin, a streptavidin molecule can also Combined with 4 biotin molecules, the affinity constant (K) of the two is also 1015mol/L. The scope of application of streptavidin is wider than that of avidin.
Application of BAS System
Biotin and Diagnostics
BAS-ELISA is a detection system established on the basis of the principle of conventional ELISA, combining the high amplification between biotin (B) and avidin (A). Biotin is easily combined with proteins (such as antibodies, etc.) by covalent bonds. In this way, the avidin molecule combined with the enzyme reacts with the biotin molecule combined with the specific antibody, which not only plays a multi-stage amplification effect, but also changes color due to the catalytic effect of the enzyme when it encounters the corresponding substrate, achieving detection. The purpose of the unknown antigen (or antibody) molecule.
The basic methods used by BAS for detection can be divided into three categories.
The first type is the labeled avidin-linked biochemical macromolecule reaction system, called the BA method, or the labeled avidin-biotin method (LAB).
The second type uses the two ends of avidin to connect the biotinylated macromolecular reaction system and the labeled biotin, which is called the bridged avidin-biotin method (BRAB).
The third type is to co-warm avidin with enzyme-labeled biotin to form avidin-biotin-peroxidase complex, and then contact with biotinylated anti-antibody, the antigen-antibody reaction system is labeled with ABC The system is integrated into one, called the ABC method. This method can amplify the signal of trace amounts of antigen thousands of times for easy detection.
Biotin and Affinity Chromatography
Affinity chromatography is to make a solid-phase adsorbent with an affinity molecule with a special structure and place it in a chromatography column. When the protein mixture to be separated passes through the chromatography column, the protein that has affinity with the adsorbent is removed. will be adsorbed and retained in the column. Those proteins with no affinity flow out directly because they are not adsorbed, so as to be separated from the separated proteins, and then select an appropriate eluent and change the binding conditions to elute the bound proteins.
Biotin-avidin systems can be combined with methods of affinity chromatography to greatly improve the purity of purified proteins, or to find receptors for known ligands. The steps are to first covalently bind biotin to the ligand protein, then add the biotinylated ligand protein to the mixture containing the receptor protein, and then pass the mixture through a pre-immobilized avidin chromatographic column. The ligand-receptor complex stays on the chromatographic column through the biotin-avidin system, and finally the receptor-ligand-protein complex or only the receptor is obtained by selective elution. This method is widely used in the drug development industry. When a drug molecule is found to be effective but it is not clear which protein it acts on, it can be biotinylated and the target protein can be extracted from thousands of proteins. “Catch” out.
The biotin-avidin system can also use a similar method to separate DNA. The method is to hang biotin on one end of the DNA probe, then use it to obtain the target DNA fragment, and then use the immobilized avidin to recover the DNA.
Biotin and localization observation
Affinity cytochemistry is a chemical reaction that utilizes the high affinity between two substances to combine with each other. In a broad sense, antigen-antibody interaction is also a kind of mutual affinity between substances. Biotin derivatives (such as biotin-conjugated lectin) are first combined with sugar chains on the cell surface, and then positioned with avidin-labeled probes. This method is more conducive to trace antigens (or antibodies) in the Localization at the cellular or subcellular level. In addition, in the process of transfection (blot technology) protein, glycoprotein or DNA, the use of biotin-avidin system-mediated staining has higher sensitivity than traditional direct staining methods.
Biotin and Gene Probes
Traditional gene probes are synthesized from radioactive phosphate bases. We can use biotin-labeled phosphate bases to synthesize gene probes to avoid possible damage caused by radioactive substances during the experiment.
Advantages of the BAS system
The great advantages of BAS in practical applications are mainly reflected in the following aspects.
Sensitivity
Biotin is easily combined with biological macromolecules such as proteins and nucleic acids, and the formed biotin derivatives not only maintain the original biological activity of macromolecular substances, but also have high specific activity and multivalent. In addition, each avidin molecule has four biotin-binding sites, which can simultaneously bind biotinylated macromolecular derivatives and labels in a multivalent fashion. Therefore, BAS has a multi-stage amplification effect, which can greatly improve the sensitivity of the detection method when applied.
Specificity
The binding between avidin and biotin has a very high affinity, and its reaction is highly specific. Therefore, the multi-level amplification of BAS improves sensitivity without increasing non-specific interference. Moreover, the BAS binding properties are not affected by high dilution of the reagents, making it possible to minimize the non-specific effects of the reagents in practical applications.
Stability
The affinity constant of avidin combined with biotin can be a million times that of the antigen-antibody reaction, and the dissociation constant of the two combined to form a complex is very small, showing irreversible reactivity; and acid, alkali, denaturant, proteolytic enzyme And organic solvents do not affect its binding. Therefore, in practical application of BAS, the stability of the product is high, which can reduce the operation error and improve the accuracy of the determination.
Universality
The versatility of the biotin-avidin system also provides a unified approach to research. For example, for a molecule to be tested, the biotin-labeled antigen for the molecule has been obtained, then the colloidal gold combined with avidin can be observed under the electron microscope, and the avidin combined with the fluorescent label can be screened by flow cytometry. With the avidin linked to the enzyme, immunohistochemical experiments such as ELISA can be performed.
Other information
BAS can be made into a variety of general-purpose reagents (such as biotinylated secondary antibodies, etc.) according to the requirements of specific experimental methods, which are suitable for different reaction systems, and can be highly diluted, the dosage is small, and the experimental cost is low; especially BAS and cost The high cost of antigen-specific primary antibody coupling can greatly reduce the amount of the latter and save the experimental cost. In addition, due to the high-speed and high-efficiency characteristics of the combination of biotin and avidin, although BAS has more reaction layers, the required incubation time is not long, and the experiment can often be completed in just a few hours.