Immunofluorescence (IF) Test

1. Dr. P. Saranraj
Head
Department of Microbiology
Sacred Heart College (Autonomous)
Tirupattur – 635 601
Tamil Nadu, India
Mobile: +91-9994146964; E.mail:
microsaranraj@gmail.com
IMMUNOFLUORESCENCE
TEST

2. IMMUNOFLUORESCENCE TEST
 In 1944, Albert Coons showed that antibodies
could be labeled with molecules that have the
property of Fluorescence.
 Fluorescent molecules absorb light of one
wavelength (excitation) and emit light of
another wavelength (emission).
 If antibody molecules are tagged with a
fluorescent dye, or fluorochrome, 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

3. Fluorescent Dyes used in
Immunofluoresecence Test
 Fluorescein - an organic dye that is the most
widely used label for Immunofluorescence
procedures, absorbs blue light (490 nm) and
emits an intense yellow - green fluorescence
(517 nm).
 Rhodamine – an organic dye, absorbs in the
yellow-green range (515 nm) and emits a deep
red fluorescence (546 nm).
 Phycoerythrin - an efficient absorber of light
(~30-fold greater than Fluorescein) and a brilliant
emitter of red fluorescence, stimulating its wide
use as a label for Immunofluorescence.

4. Direct and Indirect
Immunofluoresecence Test
 Direct Immunofluoresecence Test - Uses a
single antibody that is conjugated directly to
a Fluorescent dye (Fluorescein). The antibody
recognizes the target molecule, binds to it and
the conjugated fluorescent dye can be detected
by the Fluorescent Microscope.
 Indirect Immunofluoresecence Test - Uses two
antibodies. A Primary antibody which recognizes
the target biomolecules and binds to it and a
Secondary antibody conjugated to a Fluorescent
dye, which recognizes and binds to the primary
antibody and indirectly localizes the target for
detection by the Fluorescent microscope.

5. Direct Immunofluoresecence Assay

6. Indirect Immunofluoresecence Assay

7. Applications of Immunofluoresecence
 Immunofluorescence has been applied to
identify a number of sub-populations of
Lymphocytes, notably the CD4, CD8 and T -
cell subpopulations.
 The technique is also suitable for identifying
bacterial species, detecting Ag-Ab complexes
in autoimmune disease and detecting
complement components in tissues.
 Localization of hormones and other cellular
products stained in situ.
 Localization of antigens in tissue sections or in
sub-cellular compartments.