Immunofluorescence (IF) Test
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The document discusses the immunofluorescence test, which uses fluorescent dyes to label antibodies. In 1944, Coons discovered that antibodies could be labeled with fluorescent molecules. These fluorescently labeled antibodies can then bind to antigens and be detected by their colored light emission under microscopy. There are two types of immunofluorescence tests - direct uses a single fluorescently labeled antibody, while indirect uses an unlabeled primary antibody followed by a fluorescent secondary antibody. The test has applications in identifying lymphocyte subpopulations, bacterial species, autoimmune complexes, and localizing antigens and cellular products in tissues.
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.
- 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.