FLUORESCENT ANTIBODY TECHNIQUE

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ALT CULTURE CLUB TOPIC

FLUORESCENT ANTIBODY TECHNIQUE

 

The fluorescent antibody technique is a specialized serological procedure which consists of an antigen-antibody reaction made visible by a fluorescent dye incorporated into the system.  A substance is said to be fluorescent if it absorbs light energy of one wavelength and emits light of another wavelength.

There are two main staining techniques currently employed in fluorescence microscopy, the direct and indirect method.  Fluorescein tagged antibody, prepared by allowing the globulin fraction of high titer antiserum to read with fluorescein, is employed in both methods.  The isothiocyanate derivative of fluorescein is the most satisfactory for conjugating with the globulin. 

The direct fluorescent antibody (FA) technique is easily carried out and is the most frequently employed.  It is a direct antigen-antibody combination on a microscopic slide.  The antigen is fixed on a clean, oil free slide and the homologous, fluorescent tagged antibody added.  If there is a union between the antigen and antibody, yellow green fluorescence will be observed under the ultraviolet microscope.  When the antigen is on the surface of a bacterium the morphology of the organism is clearly defined.

The indirect fluorescent antibody technique requires more steps than the direct FA but is valuable in identifying unknown organisms as well as unknown antibodies in sera and other body fluids.  In the indirect method the untagged antibody plays a dual role -- as the antibody in the primary reaction and as the antigen in the secondary reaction.  The indirect FA method also has been used to note the presence of anti-nuclear factor (ANF) in sera of patients with lupus erythematosus (LE).  The antigens are tumor imprints (CA breast) fixed on the slide in absolute alcohol and stored at -20 C.

The FA technique is a valuable diagnostic tool because of the rapidity with which the results obtained.  In many instances results maybe reported the same day that a specimen is received in the laboratory.  However, depending on the laboratory, small or big, due to costly commercial FA kits and manpower time, all patient's serum positive for RPR card screens  are saved for FA testings.

Other past or outdated uses: Rectal swabs maybe smeared immediately on slides and examined for Escherichia coli serotypes; throat swabs that have been incubated in Todd-Hewitt broth for 2 hours may then be examined for Group A Streptococci (GAS) -- the broth is centrifuged, the sediment washed in saline and smears made and examined with direct FA method.  The FA technique also has been employed in identifying Haemophilus influenza type B, Klebsiella, Neisseria gonorrhoea, Bordetella pertussis, Brucella abortus, Leptospira, Cryptococcus, Listeria serotypes.  It has been successful in identification of antibodies to T. pallidum.

There are advantages to the FA technique other than the rapidity of obtaining results.  The fluorescein tagged antibody will react with non-viable pathogens as well as viable ones.  Organisms in a positive specimen will fluoresce even though they are intracellular.  Immuno-fluorescence reveals the presence of a small number of organisms or organisms in a contaminated culture.

There are technical complications involved in the immunofluorescence procedure.  Cross reactions, non-specific staining and autofluorescence are the major source of trouble.  Cross reactions are due to common antigens.  Non-specific staining may result in fluorescence of an antigen in the absence of specific antibodies in the conjugate.  This maybe due to unreacted fluorescent material.  Autofluorescence due to natural fluorescence of certain body tissues also may present a complication.  Another disadvantage is obtaining adequately trained personnel - if not properly instructed - leading to human error.

The following are lists of precautions and consideration when doing FA technique:

Specimen

1. 
   
   If there are too few organisms, the specimen should be centrifuged.  If too many, it should be diluted.


2. 
   
   Care must be exercised to prevent the loss of antigen from the slide during processing.  To detect this, observe the slide under tungsten light source of microscope prior to examination with UV light.


3. 
   
   If excessive amount of tissue or mucous is present, counterstain with Bacto-Rhodamine or adsorb the conjugate with animal tissue powder to reduce auto and background fluorescence

Slide

1. 
   
   The slide must be grease-free.


2. 
   
   The specimen must be adequately fixed to the slide.

Conjugate

1. 
   
   The reaction spectrum of the conjugate must be known.


2. 
   
   The titer should also be known so that it might be diluted prior to use.  Optimal dilution of the conjugate reduces the possible degree of cross-reaction, fluorescence and non-specific background fluorescence.

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