• 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
• 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
• 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.
• 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
• However, because of its carbohydrate content and basic pI, avidin exhibits
relatively high nonspecific binding properties.
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
• The primary antibody is incubated with the tissue sample to allow
binding to the target antigen.
• A biotinylated secondary antibody, with specificity against the
primary antibody, is incubated with the tissue sample to allow
binding to the primary antibody.
• 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.
• 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.
Important features of the Avidin-Biotin Complex (ABC) staining
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
Requires less primary antibody
than direct methods of detection
Reduced assay time compared
to the PAP method
• Direct method
In this method a labelled antibody (e.g. with a substrate-
chromogen) reacts directly with the antigen in the tissue.
The advantage is that only one antibody is needed,
hence the application is fast and produces little
nonspecific binding. On the other hand, since only one
antibody binds one epitope, the signal intensity is low.
For applications with small amounts of antigen the signal
may be too weak.
• Indirect method
In order to upscale the low signal intensity of the direct
method, the indirect method has been developed. The
primary antibody binds to the antigen, followed by a
second (labelled) antibody binding the primary. The
signal is amplified due to the binding of multiple
secondary antibodies to a single primary antibody.
Another advantage is that only one labelled antibody
needs to be employed for different targets, which may
reduce costs. One disadvantage is that nonspecific
binding occurs more frequently than with the direct
• PAP method
Today, the peroxidase anti-peroxidase method is rarely
used, but has been popular in pathology laboratories
previously. It is an indirect method which depends on a
third layer of antibodies, bound to peroxidase, that binds
an unconjugated second layer antibody. The peroxidase
is not chemically conjugated to the IgG but
immunologically bound. That means that the third layer
antibody is specific for peroxidase. This leads to a much
higher activity of the peroxidase, in turn increasing the
assay sensitivity by two to three orders of a magnitude.
• (Strept)Avidin-Biotin Complex (ABC) methods
Nowadays, one of the most commonly used method for
staining is based on the high affinity that avidin (chicken
egg) and streptavidin (Streptomyces avidinii) have for the
glycoprotein biotin. The basic principle is that avidin (or
streptavidin) reacts with a biotinylated secondary
antibody, followed by a horseradish peroxidase reaction.
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