Direct Fluorescent Antibody Test

Principles


Fluorescence microscopy requires a special type of light source, usually a mercury lamp. The light from the lamp passes through special colored filters which only allow light with distinct wavelengths to pass. When this narrow band of light hits the specimen, certain compounds in the specimen (either natural compounds or added fluorescent chemicals) capture the light and reflect it back up as light with a lower energy. This reflected light is detected either with the viewer's eye, or with sensitive detectors. Because this type of microscopy uses reflected light on a dark background, very small amounts of light (and of your sample) can be seen. Fluorescent compounds include natural compounds such as chlorophyll, as well as certain DNA-binding dyes such as ethidium bromide and DAPI.

The Direct Fluorescent Antibody Test detects the presence of a particular antigen (typically a specific protein on the surface of a virus, bacterium or other microbe).

Fluorescent chemicals are attached to the constant region of an antibody. If the antigen is present, the antibody binds to generate a very specific, very sensitive protein tag.

To see an animation explaining this method, click here: DFA Animation

Procedure

First you must buy or prepare a fluorescein-labeled antibody. This antibody must be specific for the organism or protein you are trying to detect.  

  1. Prepare your sample by fixing it to the slide.
  2. Add the fluorescein-labeled antibody.   Give them time to bind,
  3. Rinse off unbound antibody and observe the slide under a fluorescent microscope.   If the sample contains the antigen of interest, it will emit light.

Advantages

  • is both sensitive and specific (need mono-clonal antibodies)
  • can use on microbes that can't be easily cultured
  • can label single cells
  • 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

  • cross reactivity may be a problem-- often difficult to develop the monoclonal antibody that works well.
  • must run careful controls to assure no false positives or negatives

 

 Fluorescent in Situ Hybridization (FISH) method

 

 

Examples

individual Legionella cells labeled with FITC-tagged antibody Legionella cells imbedded in a polysaccharide biofilm layer Cryptosporidium oocysts (smaller green spheres) labeled with FITC-tagged antibody