Fluorescence antibody test

FAT technique mostly used in immunohistochemistry to identify Ag-Ab reaction.
it is a microscopic-based technique mostly uses Ag-Ab complexes for diagnosis.
this presentation will describe direct, indirect and complement indirect immunofluorescent.

1. F LUORESCENCE A NTIBODY T EST By: KINZA HAROON SAEED JAMAL

2. Immunofluorescence Immunofluorescence is a Microscopic-based technique, used clinically to diagnose certain cutaneous diseases by the detection of Antigen-Antibody complexes . Techniques including Direct, Indirect, and Complement indirect immunofluorescence are utilized depending on clinical scenario . IF studies are considering the “Gold Standard” for autoimmune blistering diseases . Examples included ; thyrotoxicosis. 2

3. Principle: Immunofluorescence is an assay which is primarily on biological samples and is classically defined as procedure to detect antigen in cellular contexts using antibodies. The specificity of antibodies to their antigen is the base for immunofluorescence. 3

4. Direct immunofluorescence: Glass slide. Substrate section. Cryostat. Phosphate-buffered saline. Moist chamber. FITC-Conjugated antibody specific to the antigen of interest. Buffered glycerin. Fluorescent microscope. 4

5. Steps: Patients sample (skin or mucosal biopsy). About 4-6ul of the snap-frozen sample is sectioned using a cryostat and placed on a glass slide, and air-dried foe 15 min. After washing by phosphate-buffered saline, the conjugated antibodies that are specific to the antigens of interest in the patient sample are added into the slide and incubated in a moist chamber. Wash, mount with glycerin, place on the cover slip, and examine under fluorescent microscope. 5

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7. Advantages of Direct Immunofluorescence: This technique is used to detect viral, parasitic, tumor antigens from patient specimens. Anatomic identification of anatomic distribution of an antigen within tissue or compartments of cell. Advantages of Direct Immunofluorescence : Shorter sample staining times. In case where one has multiple Ab raised in the same species, a direct labelling may be necessary. 7

8. Disadvantage of Direct Immunofluorescence Lower signal, Generally higher cost, Less flexibility, Difficulties with labelling procedure when commercially direct conjugates are unavailable. 8

9. 2. Indirect Immunofluorescence Reagents: Substrate section. Glass slide. Patient sample. Moist chamber. Phosphate buffered saline. FITC-Conjugated secondary antibodies specific for Fc region. Glycerin. Fluorescent microscope. 9

10. Steps: After placing the substrate section on a glass slide, add the serial diluted patient serum, and incubate in a moist for 30 min. positive and negative control sera must be used to test the antibody reactivity. Wash with phosphate-buffered saline, and add the conjugated antibodies that are specific to the human antibody Fc region. Wash for at least 10 times with phosphate-buffered, mount and examine under the fluorescent microscope. 10

11. Indirect immunofluorescence Steps involve in indirect immunofluorescence. 11

12. A pplications of Indirect Immunofluorescence: It is often used to detect autoantibodies in serum or other body fluids. Used in Dermatology primarily to detect circulating pathogenic autoantibodies. Commonly used in the detection; Anti-nuclear antibodies, Systematic lupus erythematous, Antithyroid antibodies . 12

13. Advantages of Indirect immunofluorescence: Greater sensitivity than direct immunofluorescence For every antibody there is a characteristics fluorescence pattern. Disadvantages of Indirect immunofluorescence: Potential cross reactivity. Finding labeled primary Ab which is more difficult to get especially for multiple labeling experiments. 13

14. Complement Indirect Immunofluorescence: Reagents: Tissue substrate. Glass slide. Phosphate-buffered saline. Patient sample. Heating source. Complement source such as fresh human serum. 14

15. Continue…. FITC-conjugated anti-human C3 antibodies. Glycerin. Cover slip. Fluorescent microscope. 15

16. Steps: After placing the substrate section on a glass slide, add the patient serum or plasma that was heated at 56 ºC for 30 min to destroy all the complement without affecting the antigens or antibodies present in the serum. Add the complement source. The complement system is activated by the antibodies that are bounded to the antigens on the slide, releasing numerous C3 molecules that binds to the antigen-antibody complex. After washing, add the FITC-conjugated antibodies specific to human C3. Then wash, and examine under fluorescent microscope. 16

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18. Applications of Complement Indirect Immunofluorescence: Used on tissue sections. Cultured cell lines or individual cells. To analyze the distribution of proteins, glycan's, and small biological molecules. 18

19. Result Interpretation: If no fluorescence is detected under the fluorescent microscope, it is a negative sample. According to the biding pattern of the anti-nuclear antibodies and the intensity of the fluorescence (1+, 2+,3+ or 4+), the autoimmune disease can be determined in correlation with the ELISA results for double-stranded DNA, single-stranded DNA, and histone. 19

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21. Immunofluorescence Microscope : 21

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