Immunofluorescence
Lab. 10
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Immunofluorescence
• If antibody molecules are tagged with a fluorescent dye,
immune complexes containing these fluorescently labeled
antibodies (FA) can be detected by colored light emission
when excited by light of the appropriate wavelength.
• Antibody molecules bound to antigens in cells or tissue
sections can similarly be visualized.
• The emitted light can be viewed with a fluorescence
microscope, which is equipped with an UV light source.
• In this technique, known as immunofluorescence, fluorescent
compounds such as:
 fluorescein
 rhodamine,
 and phycoerythrin,
• are coupled to Abs which then ca be detected by
fluorescence microscope
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• positive test
• negative test
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Immunofluorescence
• Fluorescent-antibody can be used for staining of
cell membrane molecules or tissue sections
• In this way, the distribution of antigen throughout
a tissue and within cells can be demonstrated.
• The method can also be used for the detection
of antibodies directed against antigens already
known to be present in a given tissue section or
cell preparation.
• There are two types of IF:
• Direct
• Indirect
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Direct immunofluorescence
• The antibody to the tissue antigen is
conjugated with the fluorochrome and
applied directly.
• For example, to show the distribution of a
thyroid autoantigen reacting with the
autoantibodies present in the serum of a
patient with Hashimoto's disease, a type
of thyroid autoimmunity.
• Isolate IgG from the patient's serum,
conjugate it with fluorescein, and apply it
to a section of human thyroid on a slide.
• When viewed in the fluorescence
microscope, the cytoplasm of the follicular
epithelial cells will be brightly stained.
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Direct Immunofluorescence
• Ab to tissue Ag is labeled with fluorochrome
Fluorochrome
Labeled Ab
Ag
Tissue Section
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Indirect immunofluorescence
• In this double-layer technique,
– the unlabeled antibody is applied directly to the tissue
substrate
– and visualized by treatment with a fluorochromeconjugated anti-immunoglobulin serum
• A number of reagents have been developed for
indirect staining.
• The most common is a fluorochrome-labeled
secondary antibody raised in one species
against antibodies of another species.
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Indirect immunofluorescence
• In this case, in order to find out whether or
not the serum of a patient has antibodies
to thyroid epithelial cells,
• First treat a thyroid section with the serum,
• wash well and then apply a fluoresceinlabeled rabbit anti-human immunoglobulin;
• If antibodies were present, there would be
staining of the thyroid epithelial cells.
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Indirect Immunofluorescence
• Ab to tissue Ag is unlabeled
• Fluorochrome-labeled anti-Ig is used to detect binding of
the first Ab.
Fluorochrome
Labeled Anti-Ig
Unlabeled
Ab
Ag
Tissue Section
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Advantages of Indirect IF
• In the first place the fluorescence is brighter than with
the direct test since several fluorescent antiimmunoglobulins bind on to each of the antibody
molecules present in the first layer.
• Second, even when many sera have to be screened for
specific antibodies it is only necessary to purchase a
single labeled reagent.
• The primary antibody does not need to be conjugated
with a fluorochrome. Because the supply of primary
antibody is often a limiting factor, indirect methods avoid
the loss of antibody that usually occurs during the
conjugation reaction.
• One can also test for complement fixation on the tissue
section by adding a mixture of the first antibody plus a
source of complement, followed by a fluorescent
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anticomplement reagent as the second layer.
Immunofluorescence
Direct
Indirect
• Fix specimen on slide
• Fix specimen on slide
• Add antibody specific
• Add antibody specific
for the desired
antigen
• Look for fluorescence
for the desired
antigen
• Add second antibody
• Look for fluorescence
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Applications: Immunofluorescence
• Immunofluorescence has been applied to
identify a number of subpopulations of
lymphocytes,
– notably the CD4+ and CD8+ T-cell subpopulations.
– The technique is also suitable for identifying bacterial
species,
– detecting Ag-Ab complexes in autoimmune disease,
– detecting complement components in tissues,
– and localizing hormones and other cellular products
stained in situ.
• A major application of the fluorescent-antibody
technique is the localization of antigens in tissue
sections or in subcellular compartments.
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Detection of Autoantibodies
• Autoimmune diseases are characterized
by antibodies directed against self
antigens.
• These autoantibodies can be detected on
human or animal tissue or cells.
• Immunofluorescence is considered as the
standard test for screening and
confirmation of autoantibodies.
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Controls
• Reagent and tissue controls are necessary for
the validation of immunofluorescence staining
results.
• Without their use, interpretation of staining
would be haphazard and the results of doubtful
value.
• More specifically, controls determine if the
staining protocols were:
–
–
–
–
followed correctly,
whether day-to-day
and worker-to-worker variations have occurred,
and that reagents remain in good working order.
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Controls
• In carrying out an immunofluorescence experiment one
has to be confident that the reaction is specific and that
the Ab is in fact binding selectively to the target Ag and
not to other components of the cell or other closely
related Ags.
• In addition if no fluorescence is observed with the probe
does this mean that the Ag is not present or it mean that
there may be a problem with preparation or with the
tissue itself?
• If the correct controls are included in the experiment we
can, with high certainty, answer these questions
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Positive and Negative Controls
• TISSUE CONTROLS can be of either negative or
positive.
• NEGATIVE TISSUE CONTROLS Specimens serving as
negative controls must be processed (fixed, embedded)
identically to the unknown, but do not contain the target
antigen.
– If you detect a signal then this suggests the problem exists within
your technique or protocol
• POSITIVE TISSUE CONTROLS Again, these controls
must be processed identically to the specimen but
contain the target antigen.
• If you detect no signal then this suggests the problem
exists within your technique or protocol.
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Antinuclear Antibodies (ANA)
How is it used?
• The ANA test is ordered to help screen for
autoimmune disorders and is most often used as
one of the tests to diagnose systemic lupus
erythematosus (SLE).
• Depending on the patient’s symptoms and the
suspected diagnosis, ANA may be ordered along
with one or more other autoantibody tests.
• Other laboratory tests associated with presence
of inflammation, such as
• erythrocyte sedimentation rate (ESR)
• and/or C-reactive protein (CRP) may also be ordered.
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When is it ordered?
• The ANA test is ordered when a patient shows
signs and symptoms that are associated with
SLE or another autoimmune disorder.
• Patients with autoimmune disorders can have a
wide variety of symptoms such as low-grade
fever, joint pain, fatigue, and/or unexplained
rashes that may change over time.
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What does the test result mean?
• ANA tests are performed using different assays
(indirect immunofluorescence microscopy or by
ELISA).
• Results are reported as a titer with a particular
type of immunofluroscence pattern (when
positive).
• Low-level titers are considered negative, while
increased titers are positive
• ANA shows up on indirect immunofluorescence
as fluorescent patterns in cells that are fixed to a
slide that is evaluated under a microscope.
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• Different patterns are associated with a variety of
autoimmune disorders.
• Some of the more common patterns include:
• Homogenous (diffuse) - associated with SLE and mixed
connective tissue disease
• Speckled - associated with SLE, Sjogren’s syndrome,
scleroderma, polymyositis, rheumatoid arthritis, and
mixed connective tissue disease
• Nucleolar - associated with scleroderma and
polymyositis
• Outline pattern (peripheral) -associated with SLE
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Patterns of ANA
diffuse staining
Peripheral pattern
speckled pattern
nucleolar pattern
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Flow Cytometer
• The fluorescent antibody techniques
described are extremely valuable
qualitative tools,
• but they do not give quantitative data.
• This shortcoming was remedied by
development of the flow cytometer, which
was designed to automate the analysis
and separation of cells stained with
fluorescent antibody.
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Flow Cytometer
• Flow cytometry uses the principles of
– light scattering ,
– light excitation,
– and emission of fluorochrome molecules
• to generate specific multi-parameter data from
particles and cells in the size range of 0.5 µm to
40 µm diameter.
• Cells are hydro-dynamically focused in a sheath
of PBS before intercepting an optimally focused
light source.
• Lasers are most often used as a light source in
flow cytometry.
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Flow Cytometer
• More sophisticated versions of the instrument are
capable of sorting populations of cells into different
containers according to their fluorescence profile.
• Flow cytometry also makes it possible to analyze cell
populations that have been labeled with two or even
three different fluorescent antibodies.
• For example, if a blood sample is reacted with a
fluorescein-tagged antibody specific for T cells, and also
with a phycoerythrin-tagged antibody specific for B cells,
the percentages of B and T cells may be determined
simultaneously with a single analysis.
• highly sophisticated versions of the flow cytometer can
even perform “five-color” analyses
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Applications: Flow Cytometry
• Characterization of cell size, complexity,
antigens
• Examples:
• diagnosis of leukemia and lymphoma
• determination of CD4/CD8 counts in
patients with HIV
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WBCs analysis
• A common clinical use is to determine the
kind and number of white blood cells in
blood samples.
• By treating appropriately processed blood
samples with a fluorescently labeled
antibody and performing flow cytometric
analysis, one can obtain the following
information:
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WBCs analysis
1. How many cells express the target antigen as
an absolute number and also as a percentage
of cells passing the beam.
– For example, if one uses a fluorescent antibody
specific for an antigen present on all T cells, it would
be possible to determine the percentage of T cells in
the total white blood cell population.
– Then, using the cell-sorting capabilities of the flow
cytometer, it would be possible to isolate the T-cell
fraction of the leukocyte population.
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WBCs analysis
2. The distribution of cells in a sample population
according to antigen densities as determined by
fluorescence intensity.
– It is thus possible to obtain a measure of the distribution of
antigen density within the population of cells that possess the
antigen.
– This is a powerful feature of the instrument, since the same type
of cell may express different levels of antigen depending upon
its developmental or physiological state.
3. The size of cells.
– This information is derived from analysis of the light-scattering
properties of members of the cell population under examination.
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labeled with fluorescein (green)
labeled with rhodamine (red)
(exciting the fluorochrome)
↓
each droplet (cell) emits
the fluorescence
(small electrical change)
each dot represents a cell
Separation of fluorochrome-labeled cells with the flow cytometer which uses a
laser beam & light detector.
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: different Ag in different cells / different levels of Ag in the same type of cell
→ fluorescence intensity / the size of cells.
SAMPLE
SHEATH
LASER
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Immunofluorescence
• Flow Cytometry
 Cells in suspension are labeld with fluorescent tag
 Cells analyzed on a flow cytometer
Flow
Tip
FL
Detector
Light
Scatter
Detector
Laser
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