Indirect Fluorescent Antibody Technique (IFAT), also referred to as indirect immunofluorescence assay (IFA), is a laboratory method used to detect and identify specific antibodies or antigens in biological samples. It is commonly employed in medical diagnostics, research, and epidemiological studies.
In this technique, a specific antigen is immobilized onto a substrate, such as a microscope slide or a multiwell plate. The sample being analyzed, which may contain patient serum or other bodily fluids, is applied onto the substrate. If the sample contains antibodies that specifically recognize the antigen, they will bind to the immobilized antigen.
To visualize the bound antibodies, a secondary antibody is used. This secondary antibody is raised in a different species, such as a goat or rabbit, and is conjugated to a fluorescent tag, such as fluorescein isothiocyanate (FITC) or rhodamine.
When the secondary antibody is applied to the substrate, it binds specifically to the human antibodies present in the sample. The fluorescent tag emits a colored glow when exposed to specific wavelengths of light, allowing for the visualization of the antibody-antigen complexes under a fluorescence microscope.
The Indirect Fluorescent Antibody Technique offers several advantages over alternative methods. It provides high sensitivity and specificity, enabling the identification of low levels of antibodies or antigens. Additionally, it permits the simultaneous detection of multiple antibodies or antigens within the same sample, owing to the availability of various secondary antibodies with different fluorescent tags.
