Indirect fluorescent antibody tests (Indirect FAT)

INDIRECT FLUORESCENT TESTS (INDIRECT FAT)

  • This test is used to detect antibodies in serum or to identify antigen in tissues or cell cultures.
  • When testing for antibody, the antigen is allowed to bind on a solid surface (e.g. impression smear on a slide).
  • This is incubated with a serum suspected of containing the antibody to that antigen, and the serum is washed off leaving only specific antibodies bound to antigen.
  • These antibodies are visualized after incubating with FITC labeled antiglobulin.
  • When the unbound antiglobulin is removed by washing and the slide examined under UV light, fluorescence indicates that antibody was present in the test serum.
  • In the indirect method of detecting antigen, the material supposed to contain the antigen (smear, cell culture etc.) is incubated with the antibody specific for the antigen and after washing it is incubated with antiglobulin or anti-antibody which is tagged with FITC dye.
  • After washing it is viewed under the fluorescence microscope to detect emission of fluorescence (the antibody added first is sometimes called primary antibody and that added next is called the secondary antibody).

FAT

FAT: Apple green fluroscence indicates positive reaction

  • Advantages of the Indirect Test:
    • Each antibody molecule binding to antigen will itself bind several antiglobulin molecules, and the fluorescence become considerably brighter than in the direct test
    • By using antiglobulin specific for each immunoglobulin class, the class of the specific antibody present in the serum can be determined.
Last modified: Tuesday, 17 April 2012, 9:51 AM