Immunofluorescence Assay (IFA)
المؤلف:
APURBA S. SASTRY , SANDHYA BHAT
المصدر:
Essentials Of Medical Microbiology 2021
الجزء والصفحة:
3rd edition , p160
2025-08-20
476
It is a technique similar to ELISA, but differs by some important features:
- Fluorescent dye is used instead of enzyme for labeling of antibody It detects cell surface antigens.
- It is also used to detect antibodies bound to cell surface antigens, unlike ELISA which detects free antigen or antibody.
Principle
Fluorescence refers to absorbing high energy-shorter wavelength ultraviolet light rays by the fluorescent compounds and in turn emitting visible light rays with a low energy-longer wavelength.
- The fluorescent dye is used to conjugate the antibody and such labeled antibody can be used to detect the antigens or antigen–antibody complexes on the cell surface
- The fluorescent compounds commonly used is fluorescein isothiocyanate (FITC).
Types
Direct Immunofluorescence Assay
Step 1: Sample containing cells carrying surface antigens is smeared on a slide
Step 2: Primary antibody specific to the antigen, tagged with fluorescent dye is added
Step 3: Slide is washed to remove the unbound antibodies and then viewed under a fluorescence microscope (Fig. 1A).
Fig1. A and B: Immunofluorescence assay: A. Direct; B. Indirect.
Indirect Immunofluorescence Assay
This detects antibodies in sample. Slides smeared with cells carrying known antigens are commercially available.
Step 1: Test serum containing primary antibody is added to the slide
Step 2: Slide is washed to remove the unbound antibodies. A secondary antibody (antihuman antibody conjugated with fluorescent dye) is added Step
3: Slide is washed and then viewed under a fluorescence microscope (Fig. 1B).
Applications: Immunofluorescence assay has various applications, such as:
- Detection of autoantibodies (e.g. antinuclear antibody) in autoimmune diseases
- Detecting microbial antigens, e.g. rabies antigen in corneal smear
- Detection of viral antigens in cell lines inoculated with the specimens.
Flow Cytometry
Flow cytometry is a laser-based technology that quantitatively analyses and separates the cells as they pass through the laser beam. Flow cytometry can be used to analyze multiple parameters of cells (e.g. leukocytes) such as cell counting, cell sorting, analysis of size, shape, granularity, DNA or RNA content of a cell, etc. Important applications include:
- CD4 T cell count in HIV infected patients
- Detection of leukocytes with specific markers for the diagnosis of various lymphomas.
الاكثر قراءة في التحليلات المرضية
اخر الاخبار
اخبار العتبة العباسية المقدسة
