Biotin is a vitamin found in all living cells that’s necessary for a multitude of biological processes. Certain plant or animal tissues such as egg yolk, blood, and the brain have abundant amounts of biotin. This vitamin is small enough that it can be conjugated to other molecules without altering their biological function. The accurate and specific interactions of biotin with biotin-binding proteins such as streptavidin and avidin make it incredibly useful when detecting and targeting biological analytes. Biotin is also a necessary ingredient when creating biotinylated antibodies.
Biotinylated antibodies are blood proteins that bind to biotin through the process of biotinylation. These antibodies are produced to respond to specific antigens; scientists use them as probes during cell research. Antibodies with specific antigen counterparts can be conjugated with biotin to form a molecule that will attach biotin to target cells. By then measuring or targeting biotin, scientists can properly identify and detect target cells through the use of biotinylated antibodies.
There are multiple protocols for the biotinylation of antibodies. Typically, they all rely on the interaction between biotin and either streptavidin or avidin. These are currently some of the strongest non-covalent interactions that can occur between proteins and ligands.
These bonds are immune to pH pressures, high salt concentrations, temperature extremes, and denaturants. This makes biotinylated antibodies useful when targeting specific biological molecules. Although used for a variety of things, one of the current practical applications for biotinylated antibodies is cell separation.
Biotinylated antibodies act as a biological marker for the identification of cell populations. Certain cell separation methods don’t have the capability to target unique cells. If those unique cells are antigen-presenting cells (APCs) with receptors on their surface, they will be able to interact and bind with specific antibodies. However, the amount of time it would take for a biotech company to develop individual products for all different cell types which need to be isolated is not conducive to efficient cell research. By making products that can track biotin and biotin-binding proteins such as streptavidin, which will conjugate with antibodies that can bind to target cells, biotech companies are effectively able to isolate a much wider range of cells.
Streptavidin is a biotin-binding protein that can be used to bind antibodies with biotin for cell separation. It’s composed of 4 identical polypeptide chains, making it a homotetramer. Subunits of streptavidin are synthesized in the form of 183 amino acid peptides. Each molar unit of streptavidin has four binding sites for biotin.
Streptavidin can be altered to make recombinant streptavidin that weighs less and amplifies the signal power of biotin-labeled cells. Streptavidin is most useful for its high affinity to biotin.
The formation of a biotin-streptavidin complex is stable over a wide range of temperatures and pH values. Due to the high affinity of these molecules, their bind is practically irreversible through physiological conditions. The measures that must be taken to reverse biotin-streptavidin binding would permanently damage the cell health and physiology of both molecules. This can be a disadvantage or an advantage depending on what you plan to use the streptavidin for. When working with cells and labeling them with no hopes of reversing the bond, the high-affinity binding is beneficial to ensuring accurate sorting results.
The process for binding streptavidin to biotin is the labeled streptavidin-biotin (LSAB) method. LSAB amplifies cell signals through the incubation of streptavidin complexes/biotinylated antibodies with reporter enzymes. Small complexes can penetrate the tissue which improves sensitivity up to eight times the sensitivity of the avidin-biotin complex method.
In LSAB, the primary antibodies are incubated alongside secondary biotinylated antibodies. Then, streptavidin that has been conjugated with enzymes is added and incubated, resulting in the occupation of the complex’s biotin-binding sites. Avidin can also be used for biotinylation in a similar manner.
When using avidin for antibody biotinylation, the avidin-biotin complex (ABC) method is used. ABC uses biotinylated antibodies to incubate avidin-biotin complexes. The complexes created in ABC are larger than those created in the LSAB method, sometimes too large to pass through tissue.
First, the primary antibody is incubated in the sample to allow binding with the desired antigen. Then, a secondary biotinylated antibody is added to the incubation with tissue to bind with the primary antibody. Avidin is then mixed with a biotinylated enzyme to make avidin-biotin-enzyme complexes.
When the avidin-biotin complexes are added to the sample they bind to the biotinylated antibodies that are attached to the tissue sample. By preparing the ABCs and target cells with the same antibodies, scientists can bind biotin to enriched samples.
Both avidin and streptavidin are biotin-binding proteins that can be used for cell identification or isolation. However, there are key differences that dictate the circumstances in which each protein should be used.
Avidin is an antibiotic for reptile, amphibian, and bird eggs. The mass of avidin from a chicken is around 67,000 to 68,000 daltons. For reference, one dalton is equivalent to the weight of a single proton, roughly 1.66 x 10-24 grams. Avidin is also highly glycosylated–about 10% of its mass is a carbohydrate, which contributes to its high solubility in water. It’s easily extracted from chicken eggs and can be chemically modified to detect biotinylated molecules in many conditions. Unfortunately, avidin results in a high amount of nonspecific binding. When using avidin for downstream assays it’s important to follow proper blocking and washing protocols.
Streptavidin is also thought to function as an antibiotic but is isolated from a bacterium named Streptomyces avidinii. The protein itself has a mass of around 60,000 daltons. Unlike avidin, streptavidin has no carbohydrates which significantly reduces its solubility in water. The lack of glycosylation also lowers nonspecific binding, making streptavidin a more efficient alternative for cell separation with the intention of downstream assays.
Biotinylated antibodies enable researchers to customize their cell separation efforts. With extra steps, proteins like avidin or streptavidin can be incubated to bind with antibodies that attach to specific tissue. Certain antibody-based methods of cell separation, such as magnetic-based cell sorting, can be limited by their biomarkers. Proteins like streptavidin increase the potential use cases for cell separation methods.
Streptavidin can be used to coat tiny glass bubbles with biotin-streptavidin complexes that bind to antigens on the surface of target cells in a sample. The strength of the streptavidin bonds mixed with the gentle nature of the bubbles allows the fast, gentle, and accurate isolation of enriched substances with a technique called BACS.
Buoyancy Activated Cell Sorting (BACS) is a cell separation technique developed by Akadeum Life Sciences that uses streptavidin as the main biotin-binding protein. This process uses bubbles to float unwanted cells to the top of a solution with antibody-based targeting. The innovative microbubble isolation kit is stirred directly into the sample container, allowing the bubbles to bind evenly to a target cell population. The bubbles will then float labeled cells to the surface of the mixture for simple collection. Once the labeled cells are removed, a high-purity enriched sample will be left untouched at the bottom of the container. These isolated cells can be used for downstream assays.
Akadeum also offers a Streptavidin Microbubbles Kit for use in targeting, capturing, and sorting cells or other analytes from biological samples.
Akadeum’s Streptavidin Microbubbles are shipped ready for biotinylated-antibody conjugation. They can also be conjugated to other biological molecules or be customized to suit your needs. Contact our scientists today for more information on how our Streptavidin Microbubbles can be catered to your specific cell separation assays.
Contact us if you’re interested in a commercial partnership, have questions, or are interested in microbubbles and how they work. For more information, download the ultimate guide to microbubble technology.