Fluroscent Antibody Test

EXERCISE-14: DIAGNOSIS OF RABIES BY FLUORESCENT ANTIBODY TEST

  • Rabies is an acute viral infection in man and warm blooded animals manifested with abnormal behavior, nervous disturbances, impairment of consciousness, ascending paralysis and death.
  • It can be diagnosed by many techniques but, the fluorescent antibody technique is the confirmative and most reliable test. Antigen can be demonstrated using the smear taken from cornea, brain (hippocampus, cerebellum and medulla) and saliva.
  • Negribodies are present in the hippocampus of carnivores and purkinji cells of herbivores rarely in the ganglionic cells of medulla, retina and salivary glands.
  • Negribodies are acidophilic matrix containing a number of minute bluish granules is oval or round structure about 2-8µ in diameter.
  • Negribodies can be detected by Seller’s staining technique.

Principle of FAT

  • Fluorescent antibody test in which fluorescently tagged antibodies bind to the appropriate antigen, causing the antigen to fluoresce, can be used to detect primary antigen-antibody reaction.
  • Organic dyes that emit absorbed light at characteristic wavelengths are called fluorochromes. Two commonly used fluorochromes are fluorescein and rhodamine. Fluorescein’s absorption maximum is at 490-495 nm; its characteristic green colour is emitted at 517 nm.Rhodamine’s absorption maximum is at 550 nm; its characteristic red colour is emitted at 580 nm.
  • To conjugate fluorochromes with antibodies, fluorochromes with chemically reactive groups are mixed with purified antibodies of the desired specificity. The two chemical forms are fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate. These compounds readily bind covalently to proteins at an alkaline pH.

Fluorescent_antibody_test

Figure: Fluorescent antibody test

Direct FAT for rabies

  • The fluorescently labeled antibody (rabies anti-nucleocapsid antibody labeled with FITC) has specificity for the antigen (rabies virus) to be detected.
  • Fluorescently tagged antibodies bind to the appropriate antigen, causing the antigen to fluoresce.

Direct_fluorescent_antibody_test

Materials required

Materials required

Quantity and purpose

Glass slide

Two numbers for taking impressin smear from infected materials

Tissue paper

To mob the cut surface of the affected organ

Phosphate buffer saline (PBS)

For washing of unbound labeled antibodies

Cold acetone in a coplin jar

For fixing the smear

Micropipette and tips

Adequate numbers 

Fluorescent (FITC) labeled rabies anti-nucleocapsid antibody

20 µL per slide

Humid box (water-soaked cotton in a box) and Incubator

For incubation of slides

10% glycerol saline, Coverslip and Fluorescent microscope

For examination of stained smear under FAT microscope

Procedure

  • Cut a portion of hippocampus major from a dog died of rabies (suspected) and mob with tissue paper.
  • Take at least three impression smears over a clean slide.
  • Fix the smear with cold acetone for 60 minutes.
  • Air-dry the slide.
  • Place 10 µl of rabies anti-nucleocapsid antibody labeled with FITC.
  • Keep the slide in a humid box and incubate at 37ºC for 60 minutes.
  • Wash the slide with PBS to remove unbound labeled antibodies.
  • Air-dry the slide.
  • Place a drop of 10% glycerol saline over the smear and put a coverslip.
  • Observe under fluorescent microscope at about 490-495 nm.
  • For reference the test must always be accompanied with positive and negative control.

Interpretation of result

  • Specific apple-green fluorescence could be seen in positive cases.

Note

  • This technique is used to identify the presence of antigen in the clinical specimens. Direct FAT is less sensitive because only one or two labeled antibodies with FITC bind to each antigenic site.
  • This level of binding leads to low fluorescence intensity.
Last modified: Wednesday, 16 May 2012, 6:31 AM