Immunochemistry is the identification of a certain antigen in a histological tissue section or cytological preparation via an antibody specific to the antigen In 1981 a new generation of immunohistochemical methods emerged with the advent of the avidin-biotin methods, which remains widely used today .All avidin-biotin methods rely on the strong affinity of avidin or streptavidin for the vitamin biotin. The two most common for amplifying the target antigen signal in IHC are called avidin-biotin complex (ABC) and labeled streptavidin binding (LSAB) Applications for which the avidin-biotin interaction is used include: Enzyme linked immunosorbent assay (ELISA) Immunohistochemistry (IHC) Western, Northern and Southern blotting Immunoprecipitation Cell-surface labeling Affinity purification Fluorescence-activated cell sorting (FACS) Electromobility shift assays (EMSA)

1. GEETHU
S NAI R
2011-09-104
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2. Immunochemistry is the identification of a
certain antigen in a histological tissue
section or cytological preparation via an
antibody specific to the antigen
The localization of the primary antibody (and
therefore the target antigen) is then
visualized microscopically via an appropriate
enzymatic or fluorescent detection system
INTRODUCTION
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3. Immunohistochemistry (IHC) combines
histological, immunological and biochemical
techniques for the identification of specific
tissue components by means of a specific
antigen/antibody reaction tagged with a
visible label. IHC makes it possible to
visualize the distribution and localization
of specific cellular components within a cell
or tissue.
INTRODUCTION
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4. The use of immunohistochemistry to study
cellular markers that define specific phenotypes
has provided important diagnostic, prognostic,
and predictive information relative to disease
status and biology.
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5. Cells to be stained can be attached to a solid
support to allow easy handling in subsequent
procedures. This can be achieved by several
methods: adherent cells may be grown on
microscope slides, coverslips, or an optically
suitable plastic support. Suspension cells can
be centrifuged onto glass slides (cytospin),
bound to solid support using chemical linkers, or
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6. I n 1981 a new generat ion of
immunohist ochemical met hods
emerged wit h t he advent of t he
avidin-biot in met hods, which
remains widely used t oday .
 All avidin-biot in met hods rely on6 07/06/14

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8. Other names
• Vitamin B7;
• Vitamin H;
• Coenzyme R;
• Biopeiderm
• The molecular formula for biotin is
CC1010 HH1616 NN22 OO33 S.S.
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9. Chemical structure of biotin.

10. • Biotin is
a coenzyme for carboxylase enzymes,
involved in the synthesis of fattY
acids, isoleucine, and valine, and
in gluconeogenesis.
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11. • D-(+)-Biotin is a cofactor responsible for carbon
dioxide transfer in several carboxylase enzymes:
– Acetyl-CoA carboxylase alpha
– Acetyl-CoA carboxylase beta
– Methylcrotonyl-CoA carboxylase
– Propionyl-CoA carboxylase
– Pyruvate carboxylase
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12. Def iciency sympt oms
include:
• Hair loss (alopecia)
• Conjunctivitis
• Dermatitis in the form of a scaly, red rash around the
eyes, nose, mouth, and genital area.
• Neurological symptoms in adults, such as depression,
lethargy, hallucination, and numbness and tingling of
the extremities.
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14. • Avidin is a tetrameric or dimeric biotin-
binding protein produced in
the oviducts of birds, reptiles and amphibia
ns and deposited in the whites of their egg
• Functional avidin is found only in raw egg, as
the biotin avidity of the protein is destroyed by
cooking
• Avidin was first discovered by Esmond
Emerson Snell (1914–2003)
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16. 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.
 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.
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18. 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 pH(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 pH and lack of carbohydrate
group.
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20. • The interaction of biotin and avidin or streptavidin has been
exploited for use in many protein and nucleic acid detection and
purification methods.
• Because the biotin label is stable and small,
• It rarely interferes with the function of labeled molecules enabling
the avidin-biotin interaction to be used for the development of
robust and
• Highly sensitive assays.

21. Schematic of the avidin-biotin
interaction. Avidin, streptavidin or
NeutrAvidin Protein can bind up to four biotin
molecules, which are normally conjugated to an
enzyme, antibody or target protein to form an
avidin-biotin complex. † denotes that avidin is
also often conjugated to an antibody, target
protein or immobilized support

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23. • 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)

24. MethodS
• The Avidin-Biotin Complex (ABC) Staining MethodThe Avidin-Biotin Complex (ABC) Staining Method
• The Labeled Streptavidin Biotin (LSAB) Staining MethodThe Labeled Streptavidin Biotin (LSAB) Staining Method

25. the avidin-Biotin coMpleX (aBc)
Staining Method

26. 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.

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29. Schematic representation of the avidin-
biotin complex (ABC) staining method.
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30. 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.
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31. 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
Important features of the Avidin-Biotin Complex (ABC) staining method.

33. THE LABELED STREPTAVIDIN BIOTIN (LSAB)
STAINING METHOD

34. • 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.

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38. Advantages of using Avidin-
Biotin Systems
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39. The avidin-biotin complex is the strongest known non-
covalent interaction (Kd = 10-15
M) between a protein and
ligand.
The bond formation between biotin and avidin is very
rapid, and once formed, is unaffected by extremes of pH,
temperature, organic solvents and other denaturing
agents.
These features of biotin and avidin – features that are
shared by streptavidin and NeutrAvidin Protein – are
useful for purifying or detecting proteins conjugated to
either component of the interaction.
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40. Like secondary antibodies, the avidin-biotin detection
system allows an almost unlimited number of primary
detection reagents (i.e., antibodies, nucleic acids probes
and ligands) to be easily captured, recovered, immobilized
or detected with a very small number of secondary
detection reagents generated by modifying avidin or
streptavidin.
 Furthermore, if a specific biotinylated molecule is not
available, there are many commercially available reagents
to facilitate biotinylation in the lab.
 Likewise, avidin and streptavidin can also be modified as
needed
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41. Disadvantages of using the
Avidin-Biotin System
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42. Although the avidin-biotin system is simple to set up and
use, it does have certain limitations.
Because any biotinylated molecule will bind to any biotin-
binding protein, these reagents must be used in
combination with other detection-probe systems (i.e.,
primary-secondary antibodies) for multiplex experiments.
Also, because biotin is a biological molecule, endogenous
biotin can cause background and specificity issues when
performing assays with certain biotin-rich tissues and
extracts (i.e., brain, liver, milk, eggs, corn).
This also applies to samples containing endogenous biotin-
binding proteins such as eggs (source of avidin) or bacteria
likeStreptomyces avidinii (source of streptavidin).
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43. For purification applications, the strength of the binding
interaction between biotin and avidin is a factor that
limits its utility. This is because harsh conditions are
required to break the avidin-biotin bonds (i.e., to
dissociate and elute), and these may denature target
proteins. To overcome this limitation, modified versions
of avidin resins and modified forms of biotin labeling
reagents are commercially available which make the
interaction readily reversible. These include monomeric
avidin, cleavable disulfide biotin reagents, and
iminobiotin and desthiobiotin derivatives (see discussion
of Protein Isolation and Enrichment below).
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44. Applications for Avidin-Biotin Probes
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45. Applications for which the avidin-biotin interaction is
used include:
Enzyme linked immunosorbent assay (ELISA)
Immunohistochemistry (IHC)
Western, Northern and Southern blotting
Immunoprecipitation
Cell-surface labeling
Affinity purification
Fluorescence-activated cell sorting (FACS)
Electromobility shift assays (EMSA)
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46. The avidin-biotin system can be used for numerous
laboratory methods. The most common methods use
avidin or streptavidin forthe detection of biotinylated
probes. The following is an overview of some of the major
laboratory methods effectively using this system.
Protein Detection
Nucleic Acid Detection
Protein Isolation and EnrichmenT
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