Avidin Biotin Methods

Avidin is a basic glycoprotein (MW 68 Kd) which has a high affinity for the small (MW 24Kd) water soluble vitamin biotin. Biotin can be conjugated to a variety of biological molecules, including antibodies, and many biotin molecules can be attached to a single molecule of protein. The biotinylated protein can thus bind to more than one molecule of avidin.

For more information, please visit our website.

However, avidin has two distinct disadvantages when used in immunocytochemical detection systems. It has a high isoelectric point of approximately 10 and is therefore positively charged at neutral pH. Consequently it may bind non specifically to negatively charged structures such as the nucleus. The second disadvantage is that avidin is a glycoprotein and reacts with molecules such as lectins via the carbohydrate moiety.

These two problems are overcome with the substitution of streptavidin for avidin. Streptavidin is a protein (MW 60 Kd) isolated from the bacterium Streptomyces avidinii , and like avidin, has four high affinity binding sites for biotin. Streptavidin has an isoelectric point close to neutral pH and therefore possess few strongly charged groups at the near neutral pH used in immunocytochemical detection systems. Furthermore streptavidin is not a glycoprotein and therefore does not bind to lectins. The physical properties of streptavidin therefore make this protein much more desirable for use in immunocytochemical detection systems than avidin.

Link to NUPTEC

The most sophisticated and sensitive extension of the streptavidin biotin technique employs pre-formed complexes. Streptavidin and biotinylated horse-radish peroxidase are simply mixed at appropriate concentrations for at least 30 minutes at room temperature for the complex to form. The pre-formed complex is then attached to the biotinylated antibody. Careful stoichiometric control ensures that some binding sites remain free to bind with biotinylated antibody. This allows the pre-formed complex to bind and provides a very high signal at the antigen binding site.

Signal Amplification

Advantages of biotinylated tyramide amplification

1. Permits the use of greatly diluted primary antibodies.
2. Enables shorter incubation periods.
3. Provides amplification of weak signals
4. Enables the demonstration of antibodies previously unreactive in paraffin sections.