IF ppt.ppt

immunoflourescence

1. 1
Immunofluorescence
Lab. 10

2. 2
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

3. 3
•positive test •negative test

4. 4
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

5. 5
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.

6. 6
Direct Immunofluorescence
•Ab to tissue Ag is labeled with fluorochrome
Ag
Fluorochrome
Labeled Ab
Tissue Section

7. 7
Indirect immunofluorescence
•In this double-layer technique,
–the unlabeled antibody is applied directly to the tissue
substrate
–and visualized by treatment with a fluorochrome-
conjugated 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.

8. 8
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 fluorescein-
labeled rabbit anti-human immunoglobulin;
•If antibodies were present, there would be
staining of the thyroid epithelial cells.

9. 9
Indirect Immunofluorescence
•Ab to tissue Ag is unlabeled
•Fluorochrome-labeled anti-Ig is used to detect binding of
the first Ab.
Ag
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled
Ab

10. 10

11. 11
Advantages of Indirect IF
•In the first place the fluorescence is brighter than with
the direct test since several fluorescent anti-
immunoglobulins 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
anticomplement reagent as the second layer.

12. 12
Immunofluorescence
•Fix specimen on slide
•Add antibodyspecific
for the desired
antigen
•Look for fluorescence
•Fix specimen on slide
•Add antibodyspecific
for the desired
antigen
•Add secondantibody
•Look for fluorescence
Direct Indirect

13. 13
•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.
Applications: Immunofluorescence