Download 7- Immunological methods

King Saud University
Riyadh
Saudi Arabia
Dr. Gihan Gawish
Assistant Professor
Dr. Gihan Gawish
Dr. Gihan Gawish
• Immunohistochemistry )IHC)
• refers to the process
proteins in cells of a
exploiting the principle
binding
specifically
biological tissues
of localizing
tissue section
of antibodies
to antigens in
• It takes its name from the roots
"immuno," in reference to antibodies
used in the procedure, and "histo,"
meaning tissue
Dr. Gihan Gawish
Immunohistochemical
staining
• Immunohistochemical staining is widely used in the
diagnosis of abnormal cells such as those found in
cancerous tumors.
• Specific molecular markers are characteristic of
particular cellular events such as proliferation or cell
death( apoptosis)
•
.IHC is also widely used in basic research to understand
the distribution and localization of biomarkers and
differentially expressed proteins in different parts of a
biological tissue.
Dr. Gihan Gawish
• Visualising
an
antibody-antigen
interaction can be accomplished in a
number of ways.
• In the most common instance, an
antibody is conjugated to an enzyme,
such as peroxidase that can catalyze a
color-producing reaction
• Alternatively, the antibody can also be
tagged to a fluorophore ,such as FITC
• FITC
is a derivative of fluorescein used in wide-ranging
applications
including flow cytometry).
Dr. Gihan
Gawish
Immunophenotyping
• Immunophenotyping is a technique used to
study the protein expressed by cells.
• This technique is commonly used in basic
science research and laboratory diagnostic
purpose.
• This can be done on tissue section (fresh or
fixed tissue), cell suspension, etc.
Dr. Gihan Gawish
Immunophenotyping
• An example is the diagnosis of leukemia .
• It involves the labeling of white blood cells
with antibodies directed against surface
proteins on their membrane.
• By choosing appropriate antibodies, the
differentiation of leukemic cells can be
accurately determined.
Dr. Gihan Gawish
Immunophenotyping
• The labeled cells are processed in a flow cytometer ,a
laser-based instrument capable of analyzing thousands
of cells per second.
• The whole procedure can be performed on cells from
the blood ,bone marrow or spinal fluid in a matter of a
few hours.
• An Example of Information provided through
Immnunophenotyping" The
flow
cytometric
immunophenotyping report indicated the malignant cells
were positive for CD19, CD10, dimCD20, CD45, HLADR, and λ immunoglobulin light chain. There was no
coexpression of CD5 or CD23 by the monoclonal B-cell
population".
Dr. Gihan Gawish
Antibody types
• The antibodies used for specific detection can
be polyclonal or monoclonal.
• Monoclonal antibodies are generally considered
to exhibit greater specificity.
• Polyclonal antibodies are made by injecting
animals with peptide antigens, and then after a
secondary immune response is stimulated,
isolating antibodies from whole serum. Thus,
polyclonal antibodies are a heterogeneous mix
of antibodies that recognize several epitopes.
Dr. Gihan Gawish
Antibody types
• Antibodies can also be classified as primary or
secondary reagents.
• Primary antibodies are raised against an antigen of
interest and are typically unconjugated (unlabelled),
• while secondary antibodies are raised against primary
antibodies.
• Hence,
secondary
antibodies
recognize
immunoglobulins of a particular species and are
conjugated to either biotin or a reporter enzyme such as
alkaline phosphatase or horseradish peroxidase (HRP).
•Dr. Gihan
Some
secondary
Gawish
fluorescent agents
antibodies
are
conjugated
to
Sample preparation
• In the procedure, depending on the purpose
and the thickness of the experimental sample,
either thin (about 4-40 μm) slices are taken of
the tissue of interest, or if the tissue is not very
thick and is penetrable it is used whole.
• The slicing is usually accomplished through the
use of a microtome, and slices are mounted on
slides.
Dr. Gihan Gawish
Immunohistochemical methods
• There are two strategies used for the
immunohistochemical
detection
of
antigens in tissue the direct method and
the indirect method.
• In both cases, the tissue is treated to
rupture the membranes, usually by using a
kind of detergent such as Triton X-100.
Dr. Gihan Gawish
Direct Method
• The direct method is a one-step staining method, and
involves a labeled antibody (e.g. FITC conjugated
antiserum) reacting directly with the antigen in tissue
sections.
•
This technique utilizes only one antibody and the
procedure is therefore simple and rapid.
•
However, it can suffer problems with sensitivity due to
little signal amplification and is in less common use than
indirect methods.
Dr. Gihan Gawish
Indirect Method
• The indirect method involves an unlabeled primary
antibody (first layer) which reacts with tissue antigen, and
a labeled secondary antibody (second layer) which reacts
with the primary antibody.
• (The secondary antibody must be against the IgG of the
animal species in which the primary antibody has been
raised.) This method is more sensitive due to signal
amplification through several secondary antibody
reactions with different antigenic sites on the primary
antibody. The second layer antibody can be labeled with a
fluorescent dye or an enzyme
Dr. Gihan Gawish
Dr. Gihan Gawish