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Avidin-Biotin Complex Method for IHC Detection

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 sites between the tetravalent avidin and biotinylated antibodies (bound to the antigen) are ideal for achieving this amplification. The two most common for amplifying the target antigen signal in IHC are called avidin-biotin complex (ABC) and labeled streptavidin binding (LSAB).

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Protein Methods Library Home

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Overview of Immunohistochemistry
(includes links to individual pages on all aspects and stages of IHC)

Biotin and Biotin-binding Proteins

Biotin

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 used to detect target antigens.

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Overview of Biotinylation

Avidin-Biotin Interactions

Avidin

The extraordinary affinity of avidin 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 68kDa. 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 formation between biotin and avidin is very rapid, 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.

Related literature...
Avidin-biotin handbook
Avidin-Biotin Technical Handbook

Streptavidin

Streptavidin is 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. Pierce products use a recombinant form of streptavidin, having a mass of 53kDa 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. 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 which binds to cell surface receptors. The RYD sequence can cause background in some applications.

IHC with streptavidin HRP
IHC using streptavidin HRP neg. control
Immunohistochemical detection of cytokeratin 18 in colon carcinoma tissue. Human colon carcinoma sections were incubated with biotinylated cytokeratin18 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 Pierce Metal Enhanced DAB.

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Avidin Conjugates

Streptavidin Conjugates

NeutrAvidin Conjugates

Deglycosylated Avidin

Thermo Scientific NeutrAvidin Protein is a specially deglycosylated version of avidin, with a mass of approximately 60kDa. As a result of carbohydrate removal, lectin-binding is reduced to undetectable levels, yet the biotin-binding affinity is retained because the carbohydrate is not necessary for this activity. NeutrAvidin Protein 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 Protein yields the lowest nonspecific binding among the known biotin-binding proteins due to its near-neutral pI and lack of both carbohydrate and RYD sequence.

Side-by-side comparison of biotin-binding proteins.
  Avidin
(chicken)
Streptavidin
(recombinant)
NeutrAvidin
(from avidin)
Molecular Weight (kDa) 67 53 60
Biotin-binding Sites 4 4 4
Isoelectric Point (pI) 10 6.8 to 7.5 6.3
Specificity low high highest
Affinity for Biotin (Kd) ~10-15M ~10-14 to -15M ~10-15M
Nonspecific Binding high low lowest

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Avidin Conjugates

Streptavidin Conjugates

NeutrAvidin Conjugates

The Avidin-Biotin Complex (ABC) Staining Method

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 information below describes the general staining procedure and a diagram of the formed complex.

  1. 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.
  2. A biotinylated secondary antibody, with specificity against 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.
  3. A biotinylated enzyme (HRP or AP) is pre-incubated with free avidin to form large avidin-biotin-enzyme complexes. Typically, the avidin and 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.
  4. 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.

Diagram of signal amplification by avidin biotin complex formation
Schematic representation of the avidin-biotin complex (ABC) staining method.

 

Important features of the Avidin-Biotin Complex (ABC) staining method.
Advantages Disadvantages
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  

 

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Immunodetection for IHC (including Substrate Selection)

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Primary Antibodies

ABC Staining Kits

Avidin Conjugates

Streptavidin Conjugates

NeutrAvidin Conjugates

Secondary Antibodies and Conjugates

The Labeled Streptavidin Biotin (LSAB) Staining Method

This 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 sensitivy of detection by 8-fold, and can be used with superior alternatives to avidin to reduce background and improve sensitivity even further. The information below describes the general staining procedure and a diagram of the formed complex.

  1. The primary and biotinylated secondary antibodies are incubated with the tissue sample as indicated in the ABC method.
  2. Enzyme-conjugated streptavidin or NeutrAvidin Protein is added to the tissue sample and incubated to allow all biotin-binding sites on the enzyme-conjugated protein to be filled.
LSAB method for IHC detection
Schematic representation of the Labeled Streptavidin Biotin (LAB) staining method.

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Primary Antibodies

Avidin Conjugates

Streptavidin Conjugates

NeutrAvidin Conjugates

Secondary Antibodies and Conjugates

Written and/or reviewed by Jared Snider, Ph.D.

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