This is pdf file for immunology

1. IMMUNOFLUORESCENCE
SUBMITTED BY
ANDAL KARTHIGA
1st Msc. MICROBIOLOGY
SRI PARAMAKALYANI COLLEGE
SUBMITTED TO
Dr.S.VISWANATHAN,Ph.D,
HEAD OF THE DEPARTMENT
SRI PARAMAKALYANI COLLEGE
SRI PARAMAKALYANI COLLEGE
( Reaccredited with A+ Grade with a CGPA of 3.9 in the III Cycle of NAAC
Affiliated to Manonmaniam Sundaranar University, Tirunelveli)
ALWARKURICHI -627 412
POST GRADUATE & RESEARCH CENTRE - DEPARTMENT OF MICROBIOLOGY
(Government Aided)

2. IMMUNOFLUORESCENCE

3. Introduction:
■ 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 specific antibodies are labelled with a compound (FITC) that
makes them glow an apple-green color when observed
microscopically under ultraviolet light.

4. History
■ Immunofluorescence studies are considered the 'gold
standard' for the diagnosis of autoimmune blistering
diseases.
■ Coons et al. (1941) developed the immunofluorescence
techniques for the first time, a discovery which made
possible to observe microscopically antigens, antibodies
and their related substances on tissue sections or on
cell smears

5. Principle
■ Immunofluorescence (IF) is a technique that permits
visualization of virtually many components in any given
tissue or cell type. This broad capability is achieved
through combinations of specific antibodies tagged with
fluorophores. Consequently, the possible applications in
research and patient care are numerous.

7. 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 - (517 nm).
green fluorescence
■ 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.

8. Examples of fluorescent dyes
Fluorescein Rhodamine

9. ■ There are two major types of immunofluorescence
staining methods:
■ 1) direct immunofluorescence: staining in which the
primary antibody is labeled with fluorescence dye,2
■ ) indirect immunofluorescence: staining in which a
secondary antibody labeled with fluorochrome is used to
recognize a primary antibody.
■ O
■ A

11. ■ Direct immunofluorescence test is used to detect unknown
antigen in a cell or tissue by employing a known labeled
antibody that interacts directly with unknown antigen. If
antigen is present, it reacts with labeled antibody and the
antibody coated antigen is observed under UV light of the
fluorescence microscope.
◗ Direct immunofluorescence test

13. Direct immunofluorescence test is widely used for detection of
bacteria, parasites, viruses, fungi, or other antigens in CSF,
blood, stool, urine, tissues, and other specimens. Few examples
include:
■ Direct immunofluorescence test for antemortem diagnosis of
rabies: The test is used for detection of rabies virus antigen in
the skin smear collected from the nape of the neck in humans
and in the saliva of dogs.

14. The need for preparation of separate labeled antibody for each
pathogen is the major disadvantage of the direct
immunofluorescence test.
■ Also used for detection of N. gonorrhoeae, C. diphtheriae, T.
pallidum, etc. directly in appropriate clinical specimens.

15.  Protocols for direct IF are usually shorter as they only
require one labeling step.
 Species cross-reactivity is minimized indirect methods as the
fluorophore is already conjugated to the primary antibody
Advantages of Direct Immunofluorescence Test

16. ■ Separately labeled antibodies need to prepared for each
pathogen.
■ Requires the use of a much more primary antibody, which is
extremely expensive.
■ Less sensitive than indirect immunofluorescence.
Disadvantages of Direct Immunofluorescence Test

17. ■ The indirect immunofluorescence test is used for detection of
specific antibodies in the serum and other body fluids for
serodiagnosis of many infectious diseases.
◗ Indirect immunofluorescence test

18. Indirect immunofluorescence is a two-stage process. In the first
stage, a known antigen is fixed on a slide. Then the patient’s
serum to be tested is applied to the slide, followed by careful
washing. If the patient’s serum contains antibody against the
antigen, it will combine with antigen on the slide. In the second
stage, the combination of antibody with antigen can be detected
by addition of a fluorescent dye-labeled antibody to human IgG,
which is examined by a fluorescence microscope.

19. The first step in the indirect immunofluorescence test is the
incubation of a fixed antigen (e.g., in a cell or tissue) with
unlabeled antibody, which becomes associated with the antigen.
Next, after careful washing, a fluorescent antibody (e.g.,
fluorescent labeled anti-IgG) is added to the smear. This second
antibody will become associated to the first, and the antigen–
antibody complex can be visualized on the fluorescence
microscope.

20. The indirect method has the advantage of using a single labeled
antiglobulin (antibody to IgG) as a “universal reagent” to detect
many different specific antigen–antibody reactions. The test is
often more sensitive than the direct immunofluorescence test.
The major limitation of immunofluorescence is that the technique
requires (a) expensive fluorescence microscope and reagents,
(b) trained personnel, and (c) have a factor of subjectivity that
may result in erroneous results.

22. ■ In detection of specific antibodies for diagnosis of syphilis,
amoebiasis, leptospirosis, toxoplasmosis, and other diseases.
■ Also used in the detection of autoantibodies that cause auto
immune disorders.
Uses of Indirect Immunofluorescence Test

23. ■ In case of secondary antibodies, a single fluorochrome-
labeled antibody is used for detecting many Ag-Ab
interactions.
■ More sensitive than direct immunofluorescence test.
■ Multiple secondary antibodies can bind to the Fc region of
primary antibody which amplifies the fluorescence signal.
Advantages of Indirect Immunofluorescence Test

24. ■ It is more complex and time-consuming than the direct IF.
■ Cross-reactivity of secondary antibody to other agents can be
problematic.
Disadvantages of Indirect Immunofluorescence Test

25. ■ Immunofluorescence can be used on tissues or cell sections
to determine presence of different biological molecules which
also includes proteins, carbohydrates, etc.
■ Also used in molecular biology for visualization of
cytoskeletons such as intermediate filaments.
■ It also plays a key role in the detection of autoimmune
disorders.
■ It can be used with some non-antibody methods of fluorescent
staining, like the use of DAPI (4′,6-diamidino-2-phenylindole )
to label DNA.
Application of Immunofluorescence

26. ■ The main problem can be photobleaching i.e. degradation of
fluorochromes.
■ It can be prevented by using higher concentration of
flurochromes and decreasing exposure time to the light.
■ Extraneous unnecessary fluorescence can occur due to
impurity of targeted antigen.
■ Autofluorescence can occur due to some agents which bear
the property of fluorescence in the given specimen.
■ It is mostly used for only fixed cells or dead cells.
■ Expensive and require higher expertise.
limitations of Immunofluorescence

27. ■ Parija S.C., (2009), Textbook of Microbiology and
Immunology, 2nd edition, Elsevier, a division of Reed Elsevier
India Private Limited, pg. 111-112.
■ Kuby immunology, 6th edition, 6th chapter, ph. 160, 161.
Reference

28. THANK YOU