Since the 1970s, biotinylation of antibodies for immunocytochemical applications has been mainly used to target antigens to cells and tissues. The general protocol for the preparation of an antibody-biotin conjugate with streptavidin was first described in 1994. It consisted of a biotinylated primary or secondary antibody applied to a protein sample. Detection was performed with labeled avidin or streptavidin. Since then, many authors have modified the method, but the basic idea remains linked to the importance of biotin-streptavidin reactions.
Biotin-streptavidin reactions are highly selective and occur rapidly when both molecules are in solution. To make a biotin-streptavidin conjugate, attach biotin to one molecule, attach streptavidin to the second molecule, and then mix the solutions.
What is biotinylation?
Biotinylation is the process of attaching biotin, a water-soluble vitamin also known as vitamin H, to a molecule (usually a protein) to either an amino acid or a carbohydrate moiety of the protein.
Avidin is a protein found in egg whites and closely related to biotin. Streptavidin is a similar protein derived from the bacterium Streptomyces avidinii, which is also closely related to biotin (source).
Biotin-streptavidin reactions
Biotin-streptavidin reactions are highly selective and rapid because of his high affinity. Many authors have described this interaction as the strongest non-covalent biological interaction, with a dissociation constant or KHey1,3×10-15M(source).
Here, K.Heyis the ligand (biotin) concentration at which half of the ligand binding sites on the protein (streptavidin) are occupied at system equilibrium and is equivalent to the following equation:
In this equation, P is the protein concentration, L is the ligand concentration, and PL is the concentration of the protein-ligand complex when the reaction reaches equilibrium. Since units of concentration are represented in moles, it stands to reason that the smaller the KHeyvalue, the greater the binding affinity of the ligand for its target (Source).
Streptavidin is a tetrameric protein and each monomer is linked to a biotin molecule. The non-covalent bonds in the binding pocket are formed by a network of hydrogen bonds between biotin and serine (SER), asparagine (ASN), tyrosine (TYR) and threonine (THR) residues, as shown in the figure below.
How to make a biotin-streptavidin conjugate
Labeled avidin binding (LAB), also called the "streptavidin binding method", was first described by Gary Bratthauer as an alternative to standard peroxidase-anti-peroxidase (PAP) or avidin-biotin complex (ABC) methods. The main differences between these protocols and LAB included: a) use of streptavidin instead of avidin because it works at neutral pH and b) higher affinity for biotin, as well as better sensitivity and resolution of the technique.
Below we discuss a general method for preparing a biotin-streptavidin conjugate. For more details read onthis article. You can also use alternative methods likeTransglutaminase bioconjugationfor protein biotinylation.
Step 1. Block slides with serum
Incubate the tissue slides overnight in 10% serum in phosphate-buffered saline (PBS). This step depends on the starting material, so the serum must be from the species from which the secondary antibody was generated.
Step 2. Add primary antibody
After removing the slides from the plate where the serum was incubated, add the primary antibody to the slide, covering it completely. Cover the liquid staining chamber and incubate for at least 30 min. Rinse with PBS, repeating three times for a few minutes between subsequent washes. See more information
Step 3. Block the slide with saline to prevent non-specific binding
Add additional 10% saline in PBS and incubate for 10 min. Rinse with PBS and repeat the procedure and incubation once more.
Step 4. Add biotinylated IgG secondary antibody.
After blotting excess serum from each slide with absorbent paper, repeat step 2 with the secondary antibody, including at least a 30 min incubation and subsequent PBS wash.
Step 5. Add labeled streptavidin.
Add labeled streptavidin (ie streptavidin-peroxidase conjugate) at an appropriate dilution such as 1:10000. Incubate for 30 minutes. Here we create the biotin-streptavidin conjugate.
Step 6. Add chromogenic solution.
Place the slides in a chromogenic solution of 3,3'diaminobenzidine tetrahydrochloride (DAB), cover and incubate for 15 min, then wash with three changes of deionized water for 2 min each. The preparation and addition of DAB as a chromogenic solution must be done with care as it is potentially carcinogenic. During the incubation you may notice a brown precipitate created by the degradation of DAB by peroxidase.
Step 7. Treat, develop blue cores, dehydrate and clean.
Counterstain with Mayer's Hematoxylin for 1-5 minutes. Rinse with deionized water, three changes, 2 minutes each. Develop blue nuclei by incubating for 10 seconds in ammonium hydroxide in water. To clean. Dehydrate with 100% ethanol, four changes of 2 min each. Clean the slides with xylene, four changes of 2 minutes each.
Etapa 8. Observe.
After you slide with Permount, look at the tissue under a microscope.
Why use an antibody-biotin conjugate?
Use an antibody-biotin conjugate as it is more specific, penetrates deeper into tissue, and is easier to obtain than antibody-protein conjugates such as antibody-HRP and antibody-AP.
Biotin-streptavidin conjugation is fast and efficient. In the above protocol, you will notice that you only need one biotinylated secondary antibody. These are very easy to find compared to HRP or AP secondary antibody conjugates. They are also smaller than HRP or AP conjugates, since biotin is a smaller molecule than HRP or AP proteins. This means that the secondary antibody can penetrate the tissue better.
Compared to Western Blotting or ELISA, using an antibody-biotin conjugate for tissue staining is better because it is difficult to find antibodies to do a Western blot or ELISA for every tissue target imaginable. ELISAs have the additional limitation of being complex to perform and have a high probability of false positives or false negatives. We delve deeper into conjugated antibodies in our article,antibody conjugation methods. If you are looking for more information about western blots, check out our articlesexposure time and western blot detectionmhow to choose Western Blot loading values.
Antibodies can be easily attached to other biomolecules such as proteins, polymers and carbohydrates using amines, carboxylates and even thiols. use theseantibody conjugation kitfor attachment of antibodies to other biomolecules.
Biotin Conjugation Applications
New biotin conjugation methods are constantly being developed toin vitromliveforms. In recent years, there have been significant advances in catalysis, cell biology and proteomics, as well as applications in the more established areas of detection, labeling and drug delivery. Below you can read about three interesting applications.
Applications of biotin conjugation include the creation of biosensors, the fluorescent labeling of living cells, and the creation of conjugated quantum dots.
Antibody-biotin-streptavidin-horseradish peroxidase (HRP) probe.
Emthis article, researchers developed a sensor to detect fumonisins (FBs), carcinogenic mycotoxins that can affect many crops. This method is based on nanomagnetic beads (NMBs) and an antibody-biotin-streptavidin-HRP system.
For this application, a FBs-specific antibody is labeled with biotin-N-hydroxysuccinimide or biotin-NHS (BNHS), and then streptavidin is used as a bridge to load HRP into the system. The NMB probe dispersed in solution competes with FBs in the sample for binding to the BNHS antibody. The streptavidin-HRP is then captured by the antibody-BNHS. Finally, HRP oxidizes the substrate and produces visible signals that can be correlated with EC concentration. Likewise, another application for a biosensor is its usehydrazine bioconjugationto detect breast cancer.
Fluorescent labeling by enzymatic biotinylation
Fluorescent labeling of live cells is a method that allows imaging of molecular events at the cell surface. To observe a fluorescent signal with any streptavidin-fluorophore conjugate, the cell surface must first be selectively biotinylated.
There are 3 common ways to biotinylate cells: Huisgen 1,3 cycloaddition, also known as click chemistry, Staudinger ligation, and enzymatic biotinylation by protein biotin ligase (BirA) produced byE.coli.
Emthis article, the researchers described the labeling of cell surfaces by enzymatic biotinylation. Here, site-specific biotinylation was performed on a 15 amino acid “acceptor peptide” (AP) by BirA. The researchers then used a streptavidin-Alexa 568 conjugate to examine the labeled proteins expressed on the surface of living HeLa cells.selective bioconjugation of methioninefor fluorescent labeling.
Dibenzocyclooctyne-poly(ethylene glycol)-biotin (DBCO-PEG-Biotin) linkers for conjugation of single domain antibodies (sdAbs).
Emthis article(sdAbs) were functionalized to target fluorescent nanocrystals (Qdots) to epidermal growth factor receptor (EGFR) positive cells. Researchers have focused primarily on nanoparticle surface engineering to enhance cell binding, with the ultimate goal of improving therapeutic delivery.
To conjugate sdAbs to the Qdot surface, they first linked the sdAbs to biotin via DBCO-PEG-biotin linkers, searching for the optimal length of the PEG chain.
They then added these functional sdAbs to a Qdot-streptavidin conjugate, looking for the surface density of Qdots—or sdAbs per Qdot—that allowed for the best cell attachment.
Antibodies can be easily attached to other biomolecules such as proteins, polymers and carbohydrates using amines, carboxylates and even thiols. use theseantibody conjugation kitfor attachment of antibodies to other biomolecules.
