Immunohistochemical staining intensity is a function of the enzyme activity, and improved sensitivity can be achieved by increasing the number of enzyme molecules bound to the tissue. The multiple binding opportunities between the tetravalent strept(avidin) and biotinylated antibodies (bound to the antigen) are ideal for achieving this amplification. The two most common methods for amplifying the target antigen signal in IHC are called the Avidin–Biotin Complex (ABC) and the Labeled Streptavidin–Biotin (LSAB) staining methods.
Biotin, also known as vitamin H, is a small molecule (MW 244.3) that is present in tiny amounts in all living cells and is critical for a number of biological processes. The valeric acid side chain of the biotin molecule can be derivatized in order to incorporate various reactive groups that are used to attach biotin to other molecules. In the context of IHC, biotin is conjugated to antibodies or to the enzyme reporters use to detect target antigens.
The extraordinary affinity of avidin (AV) for biotin allows biotin-containing molecules in a complex mixture to be specifically bound to avidin. Avidin is a glycoprotein found in the egg white and tissues of birds, reptiles and amphibia. It contains four identical subunits having a combined mass of 67 to 68 kDa. Each subunit consists of 128 amino acids and binds one molecule of biotin; thus, a total of four biotin molecules can bind to a single avidin molecule. The extent of glycosylation on avidin is very high; carbohydrates account for about 10% of the total mass of the tetramer. Avidin has a basic isoelectric point (pI) of 10 to 10.5 and is stable over a wide range of pH and temperatures. Extensive chemical modification has little effect on the activity of avidin, making it especially useful for protein purification. However, because of its carbohydrate content and basic pI, avidin exhibits relatively high nonspecific binding properties.
Avidin–biotin binding is the strongest known non-covalent interaction between a protein and ligand. The bond between biotin and avidin is formed very rapidly, and once formed, is unaffected by extremes in pH, temperature, organic solvents and other denaturing agents. These features of avidin make detecting or purifying biotin-labeled proteins or other molecules particularly useful for a number of biomedical applications.
Our 48-page Avidin–Biotin Technical Handbook brings together everything needed to biotinylate, purify or detect proteins. Featured products include cell-surface-protein biotinylation and purification kits, antibody labeling reagents, and new photoreactive biotinylation reagents. This handbook includes dozens of references along with protocols, troubleshooting tips, selections guides and a complete list of available tools.
Streptavidin (SA) is a biotin-binding protein isolated from Streptomyces avidinii, and is similar in size and affinity for biotin. In contrast to avidin, though, streptavidin is not glycosylated, which makes the protein less prone to nonspecific binding in IHC applications. Thermo Fisher Scientific products are made with a recombinant form of streptavidin, having a mass of ~53 kDa and a near-neutral pI.
There are considerable differences in the composition of avidin and streptavidin, but they are remarkably similar in other respects. Streptavidin is also a tetrameric protein, with each subunit binding one molecule of biotin with affinity similar to that of avidin. However, streptavidin is much less soluble in water than avidin. Guanidinium chloride at pH 1.5 will dissociate avidin and streptavidin into subunits, but streptavidin is more resistant to dissociation. Streptavidin contains an RYD sequence similar to the RGD sequence in other proteins that bind to cell surface receptors. The RYD sequence can cause background in some applications. In the following example, a biotinylated antibody was used to detect cytokeratin 18 in human cancer tissue.
Immunohistochemical detection of cytokeratin 18 in colon carcinoma tissue. Human colon carcinoma sections were incubated with biotinylated cytokeratin 18 antibody (top panel) or blocking buffer alone as a negative control (bottom panel). The samples were then incubated with Thermo Scientific Pierce High Sensitivity Streptavidin-HRP, and the signal was developed with Thermo Scientific Metal Enhanced DAB Substrate Kit.
De-glycosylated avidin (NeutrAvidin (NA))
Thermo Scientific NeutrAvidin (NA) Protein is a specially de-glycosylated version of avidin, with a mass of approximately 60 kDa. As a result of carbohydrate removal, lectin binding is reduced to undetectable levels, yet the biotin-binding affinity is retained because the glycosylation is not necessary for this activity. NeutrAvidin offers the advantages of a near-neutral pI (6.3) to minimize nonspecific adsorption, along with lysine residues that remain available for derivatization or conjugation. NeutrAvidin yields the lowest nonspecific binding among the known biotin-binding proteins due to its near-neutral pI, its lack of glycosylation, and the absence of the RYD sequence.
Learn more: Avidin-Biotin Interaction
|Molecular weight (kDa)||67||53||60|
|Isoelectric point (pI)||10||6.8 to 7.5||6.3|
|Affinity for biotin (Kd)||~1.3 x 10-15 M||~0.04 x 10-15 M||~1.3 x 10-15 M|
*Depending upon the application or circumstances
Reporter intensity is a function of the localized enzyme activity, and improved sensitivity can be achieved by increasing the number of enzyme molecules bound to the target antigen. The multiple biotin-binding sites in each tetravalent avidin molecule are ideal for achieving this amplification. The following information describes the general staining procedure along with a diagram of the formed complex.
- The primary antibody is incubated with the tissue sample to allow binding to the target antigen. Typical incubation times vary from 1 hour at ambient temperature to overnight at 4ºC.
- A biotinylated secondary antibody, with specificity for the primary antibody, is incubated with the tissue sample to allow binding to the primary antibody. This incubation step is usually 1 hour at room temperature but can be extended to overnight at 4ºC.
- A biotinylated enzyme (HRP or AP) is pre-incubated with free avidin to form large avidin–biotin–enzyme complexes. Typically, the avidin–biotinylated enzyme are mixed together in a specified ratio to prevent avidin saturation and incubated for about 15 minutes at room temperature to form the complex.
- An aliquot of this solution is then added to the tissue sample, and any remaining biotin-binding sites on the avidin bind to the biotinylated antibody that is already bound to the tissue.
The result is a greater concentration of enzyme (three enzyme molecules to one avidin molecule) at the antigenic site and therefore an increase in signal intensity and sensitivity upon addition of substrate.
Schematic representation of the ABC staining method.
Learn more: IHC Immunodetection
|Increased enzyme reporter localized to the target antigen||Some tissue may require endogenous biotin blocking to avoid nonspecific labeling|
|Increased detection efficiency||The ABC complex is large, which hinders tissue penetration in some applications|
|Requires less primary antibody than direct methods of detection|
|Reduced assay time compared to the PAP method|
The Labeled Streptavidin–Biotin (LSAB) staining method employs a streptavidin–enzyme conjugate to detect the bound biotinylated primary antibody on the tissue section and can be used if the avidin–biotin–enzyme complex in the ABC method becomes too large to penetrate the tissue. This smaller complex allows better tissue penetration, has been reported to improve the sensitivity of detection by as much as ~8-fold, and can be used with superior alternatives to avidin to reduce background and improve sensitivity even further. The following information describes the general staining procedure along with a diagram of the formed complex.
Schematic representation of the LSAB staining method.
Learn more: IHC Troubleshooting Guide
- Hendrickson W (1985) BioTechniques 3:346–354.
- Beisker W et al. (1987) Cytometry 8:235–239.
- Cowen T et al. (1985) Histochemistry 82:205–208.
- Mosiman VL et al. (1997) Cytometry 30:151–156.
- Romijn, Herms J et al. (1999) J Histochem Cytochem 47:229–236.
For Research Use Only. Not for use in diagnostic procedures.