BAS is a new type of biological reaction amplification system

  • Biotin-Avidin-System (BAS) is a new type of biological reaction amplification system developed in the late 1970s. With the advent of various biotin derivatives, BAS has quickly been widely used in various fields of medicine. A large number of studies in recent years have confirmed that the biotin-avidin system can almost be combined with various markers that have been successfully studied. The high-affinity and strong binding between biotin and avidin and the multi-stage amplification effect make BAS immunolabeling and related tracer analysis more sensitive. It has become a new technology widely used in qualitative and quantitative detection of trace antigens, antibodies, and localization observation.

     

    Principle

    Biotin (B) is widely distributed in animal and plant tissues, and is often extracted from high-content egg yolk and liver tissues, with a molecular weight of 244.31 Da. The biotin molecule has two ring structures. Ring I is the imidazolone ring, which is the main part for binding to avidin. Ring II is the thiophene ring. There is a valeric acid side chain on C2. The terminal carboxyl group is used to bind antibodies and others. The unique structure of biological macromolecules, after chemical modification, biotin can become a derivative with a variety of 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 or 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 after combining with biotin, the stability is better. The interaction between biotin and avidin is currently known to be the strongest non-covalent interaction. The affinity constant (K) is 1015mol/L, which is at least higher than the affinity between antigen and antibody (K=105~1011mol/L) 10,000 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 and qualitative detection of trace antigens, antibodies, and receptors, as well as localization observation research, and can also be made into affinity media for the separation and purification of reactants in the above-mentioned various reaction systems.

    Avidin has a sugar chain side chain, which makes it easy to have non-specific affinity with polysaccharides on the cell surface. Therefore, streptavidin was developed.

     

    Streptavidin (SA) is a protein secreted by Streptomyces avidinii with a molecular weight of 65kD. Streptavidin molecule is composed of 4 identical peptide chains, each of which can bind a biotin without any glycosyl, so like avidin, a streptavidin molecule can also Combining 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 diagnosis

    BAS-ELISA is based on the principle of conventional ELISA, combined with the high amplification effect between biotin (B) and avidin (A), and established a detection system. Biotin is easy to covalently bond with proteins (such as antibodies, etc.). In this way, the avidin molecule bound to the enzyme reacts with the biotin molecule bound to the specific antibody, which not only plays a multi-stage amplification effect, but also shows color due to the catalytic action of the enzyme when it encounters the corresponding substrate, so as to achieve 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 reaction system of labeled avidin linking biochemical macromolecules, called BA method, or labeled avidin biotin method (LAB).

    The second type connects both ends of avidin to the biotinylated macromolecular reaction system and labeled biotin, which is called the bridging avidin-biotin method (BRAB).

    The third type is to warm avidin and enzyme-labeled biotin to form an avidin-biotin-peroxidase complex, and then contact the biotinylated anti-antibody to label the antigen-antibody reaction system with ABC The system is connected as a whole, called the ABC method. This method can amplify the signal of the trace antigen by thousands of times for easy detection.

     

    Biotin and Affinity Chromatography

    Affinity chromatography is to make the affinity molecules with special structure into a solid phase adsorbent 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 becomes Will be adsorbed and stay in the chromatography column. Those proteins with no affinity are not adsorbed and flow out directly to separate them from the separated proteins. Then select an appropriate eluent and change the binding conditions to elute the bound proteins.

    The biotin-avidin system can be combined with affinity chromatography to greatly improve the purity of purified protein, or to find receptors for known ligands. The step is 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 chromatographic column with avidin immobilized in advance. At this time, 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 people find that a certain drug molecule has curative effects but it is not clear which protein it acts on, it can be biotinylated and the target protein can be changed from thousands of proteins. "Catch" it 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, and then use it to obtain the target DNA fragment, and then use the immobilized avidin to recover the DNA.

     

    Biotin and Positioning Observation

    Affinity cytochemistry is a chemical reaction that uses the high affinity between two substances to combine with each other. Broadly speaking, the interaction between antigen and antibody is also a kind of mutual affinity between substances. Shilling biotin derivatives (such as biotin-bound lectin) binds to sugar chains on the cell surface, and then uses avidin-labeled probes to locate, this method is more conducive to trace antigen (or antibody) in Positioning at the cellular or subcellular level. In addition, in the process of protein, glycoprotein or DNA transfection (blot technology), the use of biotin-avidin system to mediate staining has higher sensitivity than traditional direct staining methods.

    Biotin and gene probe

    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 materials during the experiment.

     

    About us

    BOC Sciences not only provides the synthesis of small molecules and biopolymers, but also contributes to functionalization or activation of compounds ready for cross-linking with pre-activated small molecules. These small molecules are either supplied by customers or prepared internally. Here are some our products, modification biology, antibody conjugation, adc technologies and antibody oligo conjugation, etc.