IF ppt.ppt
- 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 antibody specific for the desired antigen • Look for fluorescence • Fix specimen on slide • Add antibody specific for the desired antigen • Add second antibody • 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