Immunofluorescence Techniques PDF

 Is a technique allowing the visualization of a specific antigen by binding a specific antibody chemically conjugated with a fluorescence dye. are dyes which have the ability to absorb the short wavelength UV radiation and emit light of longer wave length fluoresce...

 Is a technique allowing the visualization of a specific antigen by binding a specific antibody chemically conjugated with a fluorescence dye. are dyes which have the ability to absorb the short wavelength UV radiation and emit light of longer wave length fluorescence (visible green light.) : fluorescein isothiocyanate (FITC) emits green light Tetramethylrhodamine-emits red light *This technique is sometimes used to make Viral Plaques more readily visible to the human eye. The principle of Immunofluorescence (IF) is based on the specific antigen- antibody interaction, where an antibody labeled with a fluorescent dye binds to its corresponding antigen. When exposed to ultraviolet (UV) or specific wavelengths of light, the fluorescent dye emits visible light, allowing the visualization of the antigen-antibody complexes. 1- Direct Immunofluorescence (DIF) is a laboratory technique used to detect specific antigens (proteins or other molecules) in tissue sections or cells by using a single, fluorescently labeled primary antibody that directly binds to the target antigen. 1- Sample Preparation: Tissue sections or cells are fixed onto a slide. 2- Antibody Application: A fluorescently labeled antibody specific to the target antigen is applied to the sample. 3- Incubation: The antibody binds directly to the antigen if it is present. 4- Fluorescence Detection: The slide is examined under a fluorescence microscope. The presence of fluorescence indicates a positive result, confirming the presence of the target antigen. 2- Indirect Immunofluorescence Assay (IDIFA) is a laboratory technique used to detect the presence of specific antibodies in a biological sample, typically serum. It relies on the binding of antibodies to a known antigen and the visualization of this interaction using a secondary, fluorescently labeled antibody. 1- Antigen Coating: A slide or substrate is coated with a specific antigen. 2- Patient Serum Incubation: The sample containing antibodies is applied to the slide. If the target antibodies are present, they bind to the antigen. 3- Secondary Antibody Binding: A fluorescently labeled secondary antibody that binds to the patient’s antibodies is added. 4- Fluorescence Detection: The slide is examined under a fluorescence microscope. Fluorescence indicates a positive reaction, confirming the presence of the specific antibodies. Direct IF assay (DIF) and Indirect IF Assay (IDIFA) comparison between Direct Immunofluorescence (DIF) and Indirect Immunofluorescence Assay (IDIFA) Aspect Direct Immunofluorescence (DIF) Indirect Immunofluorescence Assay (IDIFA) Detects antigens in a sample using a primary Detects antibodies in a patient’s serum by Definition antibody directly conjugated to a fluorescent binding them to antigens and using a secondary dye. fluorescent antibody. Fluorescence Label Primary antibody is fluorescently labeled. Secondary antibody is fluorescently labeled. Two steps: Antigen + primary antibody, then Steps Single step: Antigen + labeled antibody. labeled secondary antibody. Purpose Identifies antigens directly in tissue or cells. Detects antibodies present in a serum sample. Highly specific but limited to the labeled More versatile, as different primary antibodies Specificity antibody. can use the same labeled secondary antibody. Lower sensitivity due to lack of signal Higher sensitivity due to amplification from the Sensitivity amplification. secondary antibody binding. Used in pathology to detect tissue-bound Commonly used for serological tests and Applications antigens (e.g., autoantibodies in skin or kidney autoimmune or infectious disease studies. biopsies). More time-consuming and potentially more Time and Cost Faster and generally cheaper (fewer reagents). expensive (additional reagents needed).