The direct fluorescent antibody (DFA) test is a rapid microscopic procedure for detecting the presence of a particular antigen (typically a specific protein on the surface of a virus, bacterium, or other microbes) using a fluorescently labeled monoclonal antibodies (mAb). DFA technique is a valuable tool for visualizing certain bacteria and viruses that are difficult to isolate or culture from patient samples.
Principle
(Image source: Brock Biology of Microorganisms)
Fluorescent chemicals (e.g fluorescein isothiocyanate) are conjugated (attached) to the constant region of antibodies. When labeled antibodies are incubated with a test sample, the antibody will bind to an antigen, if present. Unbound antibodies are washed away. Areas, where antigens are present, are visualized as fluorescent-apple-green using a fluorescence microscope. If specific antigens are absent there will be no fluorescence.
Procedure
- Prepare the sample by fixing it to the glass slide.
- Apply an appropriate volume of specific fluorescein-labeled antibody to cover the fixed smear. Smear should not be allowed to dry during staining process.
- Incubate at 36°± 2°C for 25 to 65 minutes in a humidified chamber
- Decant excess antibody from the smear by rinsing with appropriate buffer (e.g. phosphate-buffered saline) or deionized water.
- Observe the stained smear at 100X to 200X magnification in a darkened room with the use of a UV light microscope.
Result
- Positive: Specific apple-green fluorescence
- Negative: No or little nonspecific background fluorescence
Uses of direct fluorescent antibody (DFA) test
- Identification of Legionella pneumophila and other Legionella species when isolated from the environment or patient sample.
- Detection of chlamydia elementary bodies from endocervical specimens
- Detection of respiratory syncytial virus (RSV) antigens in nasopharyngeal aspirate
- Detection of rabies virus antigen in the brain of animals suspected of having rabies infection
Advantages
- Allows visual assessment of the adequacy of a specimen
- can use on microbes that can’t be easily cultured (e.g., detection of rabies virus antigen in the brain of the animals)
- is both sensitive and specific (need mono-clonal antibodies)
- can label single cells and can view cells in natural environment
- can use different types of fluorescent-labeled antibodies, each with different dye, to see multiple cell types in one sample.
Disadvantages
- Expensive: Many individuals view the requirement for a fluorescent microscope as an expensive luxury.
- Fluorescence fades rapidly over time, which makes the archiving of slides difficult.
- It is often difficult to develop the monoclonal antibody that works well and cross-reactivity may be a problem.
References and further reading
- CDC:Direct fluorescent antibody test
- Madigan Michael T, Bender, Kelly S, Buckley, Daniel H, Sattley, W. Matthew, & Stahl, David A. (2018). Brock Biology of Microorganisms (15th Edition). Pearson.
- Fluorescent-Antibody Techniques in Diagnostic Bacteriology
- United States Department of Agriculture Center for Veterinary Biologics: Testing Protocol-Fluorescent Antibody Staining Procedure for Detection of Viral Antigens
