Immunofluorescence (IF) combines use of antibodies with fluorescence imaging techniques to visualize target proteins, antigens and other bio molecules within fixed cell or tissue samples. It is based on use of specific antibodies which have been chemically conjugated to fluorescent dyes which binds directly or indirectly to cellular antigens.
These tests are of two types:
- Direct/Primary immunofluorescence test.
- Indirect/ Secondary immunofluorescence test.
Direct immunofluorescence test
Direct immunofluorescence test is used to detect unknown antigen in a cell or tissue by employing a known labeled antibody that interacts directly with unknown antigen. If antigen is present, it reacts with labeled antibody and the antibody coated antigen is observed under UV light of fluorescence microscope.
Primary, or direct, immunofluorescence uses a single antibody that is chemically linked to a fluorophore. The antibody recognizes the target molecule and binds to it, and the fluorophore it carries can be detected via microscopy.
Steps in direct Immunofluorescence
- Fixation of antigen in slide.
- Layering of fluorescein labeled antibodies over it.
- Washing to remove unattached antibodies.
- Examination under UV light in fluorescent microscope.
The site where antibody attaches to its specific antigen will show apple green fluorescence or other colored fluorescence based on stain/ dye used.
Uses of direct Immunofluorescence
- For detection of bacteria, parasites, viruses, fungi, or other antigens in CSF, blood, stool, urine, tissues, and other specimens.
- Detection of N. gonorrhoeae, diphtheriae, T. pallidum, etc. directly in appropriate clinical specimens.
- Detection of rabies virus antigen in skin smear.
- Direct detection of Pathogens or their antigens in tissues or in pathological samples.
Advantages of direct Immunofluorescence
- Faster and easier to perform.
- Reduced non-specific background signal.
- Limited antibody cross-reactivity.
Disadvantages of direct Immunofluorescence
- Separate specific fluorescent labeled antibody has to be prepared against each antigen to be tested.
- Less sensitive and requires high amount of primary antibody.
Since the number of fluorescent molecules that can be bound to primary antibody is limited, direct immunofluorescence is less sensitive than indirect immunofluorescence.