ISSN 2398-2977      

Indirect immunofluorescence


Michael Day

Nicola Pusterla

Synonym(s): IF, IFAT


  • The indirect immunofluorescence method is used to detect serum antibodies, most often antibodies specific for an infectious agent or an autoantigen by use of an indirect immunofluorescence antibody test (IFAT).
  • ‘Indirect immunofluorescence’ is used in a different context related to the detection of an antigen in a tissue section involving a two-step procedure with a primary antigen-specific antibody that is in turn detected by a secondary antibody conjugated to a fluorochrome.
  • In contrast, ‘direct immunofluorescence’ is a one-step procedure where the primary antigen-specific antibody is directly conjugated to the fluorochrome.


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  • In the IFAT, serum from the patient is overlaid onto a substrate (eg a tissue section, an infected cell monolayer) that contains the antigen (eg infectious agent) of interest:
    • Any antibody present in the serum will bind to the antigen.
    • Antibody is subsequently detected by the use of a secondary antibody, eg rabbit anti-horse IgG, that has been conjugated to a fluorochrome.
    • Deposition of the fluorochrome-labeled secondary antibody is observed by use of the fluorescence microscope.
  • A range of serum dilutions is made (generally in the order of ½ to 1/1280) and each dilution is incubated with a separate substrate (tissue section or cell monolayer):
    • Sectored microscope slides that contain 10-12 separate replicate sections or monolayers are usually used for this purpose, so the entire assay can be performed on a single microscope slide.
    • The period of incubation varies with the test, but in general 30-60 min incubation at room temperature is sufficient to permit most serum antibodies to bind the target antigens within the substrate.
    • The slide is then washed in saline to remove unbound serum antibodies, and subsequently incubated with an antiserum directed against immunoglobulin (generally IgG, but IgM detection is important in infectious disease serology) that is conjugated to a fluorochrome (typically fluorescein isothiocyanate; FITC).
    • The conditions of incubation of the secondary reagent again vary with the particular assay. Following this incubation, the slide is again washed to remove unbound secondary antibody, mounted under an aqueous mountant and observed by fluorescence microscopy at an appropriate wavelength to permit emission of fluorescence by the fluorochrome.
  • In the case of using indirect immunofluorescence for detection of tissue antigen:
    • A section of tissue on a microscope slide is first incubated with the primary detecting antiserum and after washing unbound antiserum, the secondary fluorochrome-conjugated reagent is applied.
    • The process is similar to that described above for detection of serum antibody
    • The test is usually reported as a descriptive summary of the location and intensity of immunolabelling within the tissue.


  • IFATs for infectious disease serology are offered by specialist laboratories.
  • Very few laboratories will offer indirect immunofluorescence testing of tissue biopsy samples.



  • In such assays it is important to incorporate both positive and negative controls.
  • A negative control is generally a test in which patient serum is replaced by serum from a patient that is known to be seronegative for the antigen in question.
  • Similarly, a positive control (seropositive) serum is used in each assay.
  • In tissue indirect immunofluorescence testing, the primary detecting antiserum is replaced by an antiserum of irrelevant specificity.

Predictive value

  • Seropositive animals may have eliminated an infection immunologically or may have been successfully treated for the infection.
  • In contrast, PCR-based detection indicates the presence of microbial genetic material in the sample as evidence of current active infection.

Technique (intrinsic) limitations

  • These tests may be limited by the sensitivity and specificity, ie antigen-specific or cross-reactive, of the primary antibody.

Technician (extrinsic) limitations

  • The test is limited by availability.

Result Data

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Further Reading


Refereed Papers

  • Recent references from PubMed and VetMedResource.
  • Riveros-Pinilla D A, Leidy Acevedo G, Londono A F & Gongora A (2015) Antibodies against spotted fever group Rickettsia sp. in horses of the Colombian Orinoquia. Rev MVZ Cordoba 20, 5004-5013 VetMedResource.
  • Di Febo T, Luciani M et al (2014) Production and characterization of monoclonal antibodies against horse immunoglobulins useful for the diagnosis of equine diseases. J Immunoassay Immunochem 36 (3), 253-264 PubMed.
  • Antonello A M, Lamberti Pivoto F et al (2010) The importance of vertical transmission of Neospora sp. in naturally infected horses. Vet Parasitol 187 (3-4), 367-370 PubMed.
  • Pusterla N, Wolfensberger C, Gerber-Bretscher R & Lutz H (2010) Comparision of indirect immunofluorescence for Ehrlichia phagocytophila and Ehrlichia equi in horses. Equine Vet J 29 (6), 490-492 PubMed.
  • Gershwin L J (2005) Antinuclear antibodies in domestic animals. New York Acad Sci 1050, 364-370 PubMed.
  • Teplitsky V, Pitlik S et al (2003) Increased prevalence of Borna disease virus ELISA and immunofluorescent antibodies in horses from farms situated along the paths of migratory birds. Israel J Vet Med 58, 80-85 VetMedResource.
  • Salinas-Melendez J A, Galvan de la Garza S et al (2001) Antibody detection against Borrelia burgdorferi in horses located in the suburban areas of Monterrey, Nuevo Leon. Rev Latin Am Microbiol 43 (4), 161-164 PubMed.
  • Carter S D, Osborne A C, May S A & Bennett D (1995) Rheumatoid factor, anti-heat shock protein (65kDa) antibodies and antinuclear antibodies in equine joint diseases. Equine Vet J 27, 288-295 WileyOnline.
  • Ristic M, Holland C J, Dawson J E, Sessions J & Palmer J (1986) Diagnosis of equine monocytic ehrlichiosis (Potomac horse fever) by indirect immunofluorescence. JAVMA 189 (1), 39-46 PubMed.

Other sources of information

  • Day M J & Schultz R D (2014) Veterinary Immunology: Principles and Practice. 2nd edn. CRC Press, USA.
  • Coleto L (1999) Equine Babesiosis: A Disease Linked to Extensive Horse Raising in the Pastureland of Extremadura (‘dehesa’). In: Dynamics and Sustainability of Mediterranean Pastoral Systems. Ed: Etienne M. CIHEAM, Spain. pp 273-276.


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