IMMUNOHISTOCHEMISTRY

Immunohistochemistry
(IHC)/immunocytochemistry
(ICC) is the technique to visualize recognition
of antigen present in the tissue with the
help of corresponding antibody.
 IMMUNOHISTOCHEMISTRY

Subsequently the
Taylor and Burns in 1974 development of
monoclonal antibody
introduced a new era in the
Nakane and Pierce in 1967 IHC technique was immunohistochemistry
successfully introduced in
routine formalin-fixed
The antibody paraffin-embedded (FFPE)
conjugated with enzyme section.
acid phosphatase and
Coons et al in 1941 horseradish peroxidase.
First time applied
immunofluorescence
technique on the frozen
section by using
fluorescence labelled
antibodies.
 BASIC PRINCIPLES
The basic principle of immunohistochemistry is to demonstrate the specific
antigen in the cell by applying the corresponding antibody to have antigen-
antibody reaction.
This antigen-antibody reaction is further visualized by attaching certain label
to the primary or secondary antibody.
 BASIC PRINCIPLES

• The antigen contains an epitope or antigenic

determinant site that evokes specific
immunologic response to develop antibody.
• The antigen epitope site and antibody-binding
site have complementary geometrical and
chemical features.
• This is responsible for the antigen-antibody
reaction.
 Antigen
Antibody
Sensitivity (Immuno
globulin)

Basic
Antibody Hybridoma
Specificity Immun Technique
ology

Polyclonal
Avidity Antibody

Affinity
 BASIC IMMUNOLOGY OF IHC

ANTIGEN
 Any substance that is capable of producing an immunogenic response is
called as antigen.
 There are specific set of chemical components that evoke immunogenic
response of the antigen which is known as epitope or antigenic
determinant site.
BASIC IMMUNOLOGY OF IHC

ANTIBODY (IMMUNOGLOBULIN)
 The antibody is produced by plasma cells in response to antigenic
stimulation.
 Immunoglobulin has specific affinity against the epitope of the
antigen.
 BASIC IMMUNOLOGY OF IHC

ANTIBODY (IMMUNOGLOBULIN) κ (kappa)

Light chains
polypeptides λ (lambda)

IMMUNOGLOBULIN α (alpha)

γ (gamma)
Heavy chains δ (delta)
polypeptides
ε (epsilon)

μ (mu)

 Depending on the nature of heavy chain, immunoglobulins are labelled as IgA, IgG, IgD, IgE and IgM.
BASIC IMMUNOLOGY OF IHC

ANTIBODY (IMMUNOGLOBULIN)
 BASIC IMMUNOLOGY OF IHC

HYBRIDOMA TECHNIQUE
 In this technique abundant unlimited amount of pure homogenous
immunoglobulin is produced.
 The antibody producing B lymphocyte is fused with a malignant immortal
plasma cell (myeloma NS-1)
 The resultant hybrid cell acquires the capability of unlimited proliferation
and production of specific antibody.
 Each hybridoma cell produces only a specific antibody for a specific antigen.
 BASIC IMMUNOLOGY OF IHC

HYBRIDOMA TECHNIQUE

 The resultant antibody in this condition is known as “monoclonal

antibody”.
 “Monoclonal” produced from a single clone of cell.
 Monoclonal antibodies are homogeneous antibodies, that have specific
properties because they can bind to 1 epitope antigen and can be made
in unlimited quantities.
HYBRIDOMA TECHNIQUE
Georges Kohler dan Cesar Milstein pada tahun 1975

1) Immunisation of a mouse
2) Isolation of B cells from the spleen
3) Cultivation of myeloma cells
4) Fusion of myeloma and B cells
5) Separation of cell lines
6) Screening of suitable cell lines
7) in vitro (a) or in vivo (b) multiplication
8) Harvesting
APLICATIONS OF ANTIBODY MONOCLONAL

1. Inductions of passive immunization

2. Imaging Diagnostic
3. Molecular Diagnosis
4. Monitoring Drug Therapy
5. Drug Delivery System (DDS)
6. Isolation or Purification of new drugs
7. Cancer Therapy
BASIC IMMUNOLOGY OF IHC

POLYCLONAL ANTIBODY

 Polyclonal antibodies are heterogenous antibody mixtures that

bind to various apitops of the same antigen.
 Polyclonal antibody is generated from the different B lymphocytes
in response to the different epitopes of a single antigen.
 As they are generated from the different clone of B cells, these
antibodies are known as polyclonal.
 There is a chance of batch-to-batch variation in case of polyclonal
antibody.
POLYCLONAL ANTIBODY
BASIC IMMUNOLOGY OF IHC
ANTIGEN-ANTIBODY BINDING

Union of antigen and antibody requires:

Affinity
Avidity

Affinity and avidity determined by law of mass action

 BASIC IMMUNOLOGY OF IHC

AFFINITY

 Affinity represents the strength of the binding

capacity of the antigenic epitope with the
corresponding site of the antibody.
 It is actually the three-dimensional fit of the
epitope site of the antigen with antibody.
 BASIC IMMUNOLOGY OF IHC

AFFINITY
 Antibody affinity is the strength of the reaction between a single antigenic
determinant and a single combining site on antibody.
 It is the sum of the attractive and repulsive forces operating between the
antigenic determinant and combining site of the antibody.
BASIC IMMUNOLOGY OF IHC

AVIDITY

 Avidity means the overall functional strength of

binding capacity of antibody and antigen complex.
 The polyclonal antibody reacts with multiple
epitope sites of the antigen, and therefore the
overall strength of antigen-antibody complex is
strong.
BASIC IMMUNOLOGY OF IHC

AVIDITY
The more valency of the
Valency antibody, the greater is the
avidity.

The affinity between the individual

The avidity of an
Affinity epitope of the antigen and the
antibody depends on:
corresponding antigen-binding site of
the antibody.

Structural Three-dimensional structural

arrangement arrangement of antigen and antibody.
 BASIC IMMUNOLOGY OF IHC

TWO IMPORTANT PARAMETER:

The ability of test to detect In highly specific test, false

SPECIFICITY reaction between homologous positive reaction are absent
antigen and antibody only, and or minimal
with no other

The ability of test to detect When the test is highly

SENSITIVITY even very minute quantities of sensitive, false negative results
antigen or antibody maybe absent or minimal
 BASIC IMMUNOLOGY OF IHC

ANTIBODY SPECIFICITY

 The antibody specificity indicates the

precise detection of specific epitope of the
antigen by the antibody.
 A particular antigenic determinant site
(epitope) can be present in more than one
antigen, and therefore a single antibody
may react with different antigens.
 BASIC IMMUNOLOGY OF IHC

SENSITIVITY

 This is related to the detection of the relative amount of antigen by the

antibody in a particular technique.
 The highly sensitive technique detects low amount of antigen, and the
relative intensity of the signal of antigen-antibody reaction is much
strong.
 REFERENCE

 Dey P. Basic and Advanced Laboratory Technique in Histopathology and Cytology.

Singapore: Springer. 2018.
 Cartun R, et al. Diagnostic Immunohistochemistry: Techniques of
Immunohistochemistry. Philadelphia: Elsevier. 2018.
 Duraiyan J, et al. Aplication Of Immunohistochemistry. J Pharm Bioallied Sci. 2012
Aug; 4(Suppl 2): S307–S309
 Hnasko RM, Stanker LH. Hybridoma Technology. Methods Mol Biol. 2015;1318:15-28.
doi: 10.1007/978-1-4939-2742-5_2
 https://www.proteinatlas.org/learn/method/immunohistochemistry
THANK YOU