Immunofluorescence is a technique that utilizes fluorescent-labeled antibodies to detect specific target antigens. It is based on use of specific antibodies which have been chemically conjugated to fluorescent dyes which binds directly or indirectly to cellular antigens.
Immunofluorescence (IF) combines use of antibodies with fluorescence imaging techniques to visualize target proteins and other biomolecules within fixed cell or tissue samples. .Immunofluorescent labeled tissue sections are studied using a fluorescence microscope.
Albert Coons discovered that antibodies could be labeled with molecules with property of fluorescence i.e. fluorescent molecules which absorb light of one wavelength (excitation) and emit light of another wavelength (emission).
Principle of Immunofluorescence
Specificity of antibodies to their antigen is the base for immunofluorescence. If antibody molecules are tagged with a fluorescent dye, or fluorochrome, immune complexes containing these fluorescent labeled antibodies (FA) can be detected by colored light emission when excited by light of appropriate wavelength. Antibody molecules bound to antigens in cells or tissue sections can similarly be visualized. The emitted light can be viewed with a fluorescence microscope, which is equipped with a UV light source.
Dyes in Immunofluorescence
In this technique, fluorescent compounds such as fluorescein and rhodamine are in common use, but other highly fluorescent substances such as phycoerythrin are also routinely used.
- Fluorescein, an organic dye is most widely used label for immunofluorescence methods, absorbs blue light (490 nm) and emits an intense yellow-green fluorescence (517 nm) under UV light. It can be tagged to immunoglobulin molecules.
- Rhodamine, another organic dye, absorbs yellow-green range (515 nm) and emits a deep red fluorescence (546 nm). They are generally toxic in nature.
- Phycoerythrinis, red protein-pigment complex is an efficient absorber of light (~30-fold greater than fluorescein) and a brilliant emitter of red fluorescence which stimulate its wide use as a label for immunofluorescence. It is obtained from algae.
These molecules can be conjugated to the Fc region of an antibody molecule without affecting specificity of the antibody.
Uses of immunofluorescence
Some uses of immunofluorescence includes:
- To analyze distribution of proteins, glycans, and small biological and non-biological molecules.
- Sometimes it is used to make viral plaques more readily visible to human eye.
- Detection of pathogens and its antigens in pathological samples.
- Diagnosis of various diseases.
Immunofluorescence tests are of two types: