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First Year: Semester – II

TOPIC

Immunofluorescence

Immunotechnolgy
(18MICCC6)
S. DILSATH SULTHANA
20MIC2506
I – M.Sc., MICROBIOLOGY
BHARATHIDASAN UNIVERSITY
TRICHY – 24
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Immunofluorescence (fluorescent antibody technique)

Immunofluorescence (IF) is a common laboratory technique, which is based on the use

of specific antibodies which have been chemically conjugated to fluorescent dyes.

The method that enables antibody to emit light after conjugating with antigen is called
Immunofluorescence.

This technique was originally demonstrated by Coons et al. and Kaplan in (1942)
and (1944). They find wide spread applications in research and clinical diagnosis.

In this technique fluorescently labeled antibodies are used to detect or identify the
antigen. It is also used to detect the microorganisms present in clinical specimens
and specific antibody present in the serum.

If antibodies bind to the cells or tissues it can be observed by tagging the antibody
with a fluorescent dyes such as Fluorescein isothiocyanate and Rhodamine.

Fluorescent dyes
 The common fluorescent dyes coupled are Fluorescein isothiocyanate and
Rhodamine b.
 Fluorescein isothiocyanate (FITC) absorb blue light at wavelength 492 - 630 nm
and emit yellowish green or Apple green light with 515 - 517 nm.
 Lissamine rhodamine (LRA) or Rhodamine B absorbs yellow-green (515 nm) and
emit Orange light (546 nm).
 These dyes are conjugated to the Fc region of an antibody without affecting its
specificity. The FA technique is very useful in testing for Rabies within a few
hours with 100% accuracy.
 The fluorescent dye is subjected to short-wavelength, high energy light, which is
absorbed and emitted as light of a different wavelength. The emitted
fluorescence has a lower energy than the absorbed light, so the wavelength of
the emitted light is longer than that of the excitation light. The fluorescence can
be visualized using fluorescence microscopy.
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Principle for Immunofluorescence

The specificity of antibodies to their antigen is the base for immunofluorescence.

Immunofluorescence is a technique where antigen expression can be detected by

binding to its antibody of which the Fc region is tagged with a fluorescent dye.

These dyes are also called as fluorochromes. These dyes can conjugate with Fc region
of antibody without affecting the specificity and make the antibody fluorescent when
exposed to UV light.

These dyes can absorb a light at a particular wavelength called as excitation

wavelength and emit light at other wavelength called as emission wavelength.

This florochromes tagged with antibodies when excited with the light of appropriate
wavelength can be detected by their respective emission wavelength under a
fluorescent microscope.

Such antibodies on binding to cells or tissues expressing antigen, the specificity of the
antigen antibody binding ensures detection of fluorescence.

Immunofluorescence finds application in detecting the location and relative

abundance of a protein.

An immunofluorescence experiment is based on the following principal steps:

1. Specific antibodies bind to the protein of interest.

2. Fluorescent dyes are coupled to these immune complexes in order to visualize the
protein of interest using microscopy.
3. The effective application of this method comprises several considerations, including
the nature of the antigen, specificity and sensitivity of the primary antibody,
properties of the fluorescent label, permeabilization and fixation technique of the
sample, and fluorescence imaging of the cell.

1. Direct Immunofluorescence / IF/ ICC principle diagram

2. Indirect Immunofluorescence / IF/ ICC principle diagram

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Types of immunofluorescence
Immunofluorescence (IF) microscopy is a widely used example of immunostaining and
is a form of immunohistochemistry. Immunofluorescence (IF) or cell imaging
techniques rely on the use of antibodies to label a specific target antigen with a
fluorescent dye (also called fluorophores or fluorochromes) such
as fluorescein isothiocyanate (FITC). The fluorophore allows visualization of the
location of target distribution in the sample under a fluorescent microscope
(eg epifluorescence and confocal microscopes).

We distinguish between two IF methods depending on whether the fluorophore is

conjugated to the primary or the secondary antibody: They are direct
immunofluorescence and indirect immunofluorescence.

 Direct IF uses a single antibody directed against the target of interest.

The primary antibody is directly conjugated to a fluorophore.

 Indirect IF uses two antibodies. The primary antibody is unconjugated and a

fluorophore-conjugated secondary antibody directed against the primary
antibody is used for detection.

Protocol
For indirect immunofluorescence assay, the protocol mainly include tissue or cell
preparation, tissue or cell fixation, serum blocking, primary antibody incubation,
marked second antibody incubation, staining, result judgment and imaging. For direct
immunofluorescence assay, there are only marked primary antibody been incubated
without second antibody and other steps are same.

Direct immunofluorescence
 Primary, or direct, immunofluorescence uses a single antibody that is
conjugated directly to a fluorescent dye. [fluorophore]
 The antibody specific to the antigen is directly tagged with the Flurochrome.
The antibody recognizes the target molecule, binds to it, and the conjugated
fluorescent dye can be detected using a microscope.
 Direct FA techniques are used to identify microorganisms present in clinical
specimen.

Procedure
In this method the target antigen is coated on the glass slide. Then the anti serum is
labeled with the fluorescent dye. Then the labeled antiserum is dropped on the antigen
coated glass slide. The conjugated antibodies with fluorescent dyes are allowed to
react with antigens fixed on slide and washed to remove the unbound antibody and
incubated for few minutes.
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Then the slide is examined under fluorescent microscope, the antibody binding to
specific antigen will a emit fluorescence when observed under UV indicating presence
or absence of antigen.

Advantages:
1. This technique has several advantages over the secondary (or indirect) protocol
because of the direct conjugation of the antibody to the fluorophore.

2. This technique reduces the number of steps in the staining procedure (making it
faster) allowing for easy handling and quick visualization.

3. Helps avoid issues with antibody cross-reactivity or non-specificity which can lead
to increased background signal.

4. Lower signal strength

Disadvantages
1. However, this method lacks any signal amplification inherent for the indirect
method [Lower signal]
2. Generally higher cost
3. Less flexibility and difficulties with labeling procedure when commercially labeled
direct conjugates are unavailable.
4. Requires laborious efforts by the scientist to conjugate potential numerous different
primary antibodies required.
5. However, the number of fluorescent molecules that can be bound to the primary
antibody is limited; direct immunofluorescence is less sensitive than indirect
immunofluorescence.

Uses
This method is useful for the identification of bacteria, viruses and other Antigens.
This technique is used to identify rabies virus, antigens of a group A Streptococci ,
Enteropathogenic Escherichia coli (EPEC), Salmonella typhi, Haemophilus influenzae B,
Neisseria meningitis, Shigella sonnei, Listeria monocytogenes, autoantibody in
Pernicious anaemia.

Indirect immunofluorescence [Secondary (indirect)]

Secondary, or indirect, immunofluorescence uses two antibodies: a primary
antibody, which recognizes the target biomolecule and binds to it, i.e., the antibody
specific for the antigen called the primary antibody is unlabelled.

And a secondary antibody conjugated to a fluorescent dye, which recognizes and

binds to the constant portion of the primary antibody and indirectly localizes the
target for detection by the microscope.
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Multiple secondary antibodies can bind a single primary antibody. This provides signal
amplification by increasing the number of fluorophore molecules per antigen.

Procedure
In this method the target antigen is coated on the glass slide and then the test serum
is added containing the specific antibodies that react with the antigen and forms a
complex. After washing the slide with water it is kept in a solution containing
fluorescein labeled anti - human immunoglobulin (antibody). Then the slide is
incubated and washed. The slide is examined the under a fluorescent microscope.
The development of fluorescence confirms the presence of antibody specific to the
antigen fixed on the slide.

Uses
It is useful for detection and identification of a different lymphocyte population,
complement proteins in tissues, hormones, Treponema palladium and many bacterial
species.

It is useful for detection of specific antibodies in the serum formed by the

microorganism.

Advantages
1. Primary antibody need not to be conjugated with Flurochrome.
2. Increased sensitivity than direct method.
3. More flexible and easy labeling procedure results in greater signal detection through
amplification.
4. It is easier in that secondary antibody conjugates are commercially available.

Disadvantages
1. Potential for Cross reactivity and the need to find the primary antibody that are not
raised in the same species or of different types of isotypes when performing multiple
labeling experiments.
2. Samples with the endogenous immunoglobulin exhibit a high background
3. While this protocol is more complex than the direct method
4. Although the second approach is more time-consuming than direct
Immunofluorescence.
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Comparison of two different types of a staining for immunofluorescence:
Direct
Time Protocols for direct IF are
usually shorter as they
only require one labeling
step.
Cost Conjugated primary
antibodies are usually
more expensive than their
unconjugated
counterparts.
Complexity Fewer steps in the protocol
simplify direct methods.
Flexibility Commercially available
pre-conjugated primary
antibodies limit your
flexibility.
Indirect
The fact that you have to
use a conjugated secondary
antibody to detect the
primary antibody results in
additional steps.
Secondary antibodies are
relatively inexpensive
compared to primary
antibodies. Further cost
savings may be made by
using the same conjugated
secondary antibody to detect
different primary antibodies.
Added complexity in indirect
methods may result from
having to select the
appropriate secondary
antibody. This is particularly
relevant in multiplex
experiments where several
secondary antibodies, each
targeting a different species
and conjugated to different
dyes, are needed.
The possibility of using
different conjugated
secondary antibodies adds
greater flexibility.
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Sensitivity The signal obtained in Several secondary antibodies

direct methods may seem will bind to the primary
weak when compared to antibody resulting in an
indirect methods as signal amplified signal.
amplification provided by
the use of secondary
antibodies does not occur.

Species cross-reactivity Species cross-reactivity is Secondary antibodies may

minimized in direct
methods as the
fluorophore is already
conjugated to the primary
antibody.
cross-react with species
other than the target. The
use of pre-adsorbed
secondary antibodies can
prevent cross-reactivity.

Background Non-specific binding is Samples with endogenous

reduced through the use of immunoglobulins may
conjugated primary exhibit a high background
antibodies. with indirect methods.

Direct and indirect methods are not limited to immunofluorescence. They are also
relevant to other techniques that rely on the use of fluorophore-conjugated antibodies
such as flow cytometry, ELISA, western blot and immunohistochemistry.

Applications in biology
 Applied to detect specific proteins in cell suspension, cultured cells, tissues
sections or individual cells that are fixed by a variety of methods etc., for their
expression and location, making immunofluorescence indispensable for
scientists to solve many cell biological questions.
 Promising tool for Rapid Diagnostic methods for infectious diseases.
 It was first utilized to diagnosis influenza, later found application in detection of
other viral diseases like rabies smallpox etc.,
 Direct Immunofluorescence has been used for localization of Ig G in immune
Complexes of skin biopsies from patients suffering from systemic lupus
erythematosus. (SLE )
 Used in identifying CD4 + and CD8 + population in T cells.
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 Used in identifying bacterial species

 Detecting complement compounds in tissues
 Detecting local hormones
 Detection of presence or absence of specific DNA sequences on chromosomes.
 Immunofluorescence (IF) is a powerful approach for getting insight into cellular
structures and processes using microscopy.
 A procedure to detect antigens in cellular contexts using antibodies.
 Immunofluorescence may be used to analyze the distribution of proteins,
glycans [glycoprotein], and other antigen targets including small biological and
non-biological molecules.
 Immunofluorescence has been widely used in biological research and medical
research yield and becomes one most important and effective method
 In cases where an immunopositive signal is found, immunofluorescence also
allows researchers to determine which subcellular compartments are
expressing the antigen.
 IF staining offers the unique possibility of revealing molecules in their “native”
state, minimizing potential perturbations of protein conformation, localization
and/or function, which can occur when using fluorescence protein tagging.

Reference
https://www.abcam.com/secondary-antibodies/direct-vs-indirect-
immunofluorescence
https://www.svarlifescience.com/knowledge/technologies/immunofluorescence
https://oni.bio/nanoimager/super-resolution-
microscopy/immunofluorescence/
https://www.sinobiological.com/category/principle-of-
if#:~:text=Immunofluorescence%20is%20an%20assay%20which,samples%20in
clude%20tissue%20and%20cells.
https://ibidi.com/content/364-the-principle-of-immunofluorescence-assays
Immunology and Immunotechnology - Shyamasree Ghosh
Textbook of immunology - C. Rajendra Sagar & A. Sadguna