Direct Fluorescent Antibody (DFA) test

Principle, Procedure and Uses

Last updated on May 30th, 2021

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.


Fluorescent antibody methods for detection of microbial surface antigens
Fluorescent antibody methods for detection of microbial surface antigens
(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.

Direct fluorescent antibody (DFA) test (Image source: CDC)
DFA Procedure (Source: Cornell University)


  1. Prepare the sample by fixing it to the glass slide.
  2. Apply an appropriate volume of specific fluorescein-labeled antibody to cover the fixed smear. Smear should not be allowed to dry during staining process.
  3. Incubate at 36°± 2°C for 25 to 65 minutes in a humidified chamber
  4. Decant excess antibody from the smear by rinsing with appropriate buffer (e.g. phosphate-buffered saline) or deionized water.
  5. Observe the stained smear at 100X to 200X magnification in a darkened room with the use of a UV light microscope.


  • Positive: Specific apple-green fluorescence
  • Negative: No or little nonspecific background fluorescence

Uses of direct fluorescent antibody (DFA) test

  1. Identification of Legionella pneumophila and other Legionella species when isolated from the environment or patient sample.
  2. Detection of chlamydia elementary bodies from endocervical specimens
  3. Detection of respiratory syncytial virus (RSV) antigens in nasopharyngeal aspirate
  4. Detection of rabies virus antigen in the brain of animals suspected of having rabies infection


  1. Allows visual assessment of the adequacy of a specimen
  2. can use on microbes that can’t be easily cultured (e.g., detection of rabies virus antigen in the brain of the animals)
  3. is both sensitive and specific (need mono-clonal antibodies)
  4. can label single cells and can view cells in natural environment
  5. can use different types of fluorescent-labeled antibodies, each with different dye, to see multiple cell types in one sample.


  1. Expensive: Many individuals view the requirement for a fluorescent microscope as an expensive luxury.
  2. Fluorescence fades rapidly over time, which makes the archiving of slides difficult.
  3. It is often difficult to develop the monoclonal antibody that works well and cross-reactivity may be a problem.

References and further reading

About Acharya Tankeshwar 474 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.

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