ISSN 2398-2942      

Immunofluorescent antibody tests


Paul Burr

Michael Day

Synonym(s): IFA; IFAT


  • Immunofluorescent antibody tests are most commonly used to detect antibodies in serum or other body fluids, most often antibodies specific for an infectious agent or an autoantigen.
  • Sample from the patient is overlaid onto a substrate, such as a tissue section or infected monolayer that contains the antigen of interest.
  • Antibody present in the serum will bind to the antigen.
  • This antigen-bound antibody is subsequently detected by the use of a secondary antibody that has been conjugated to a fluorochrome.
  • Deposition of the fluorochrome is observed by use of the fluorescence microscope.
  • Similar methodology is used to detect infected cells in blood or aspirated fluid, eg FeLV infected cells in blood smears. In this case a smear is prepared from the sample and infected cells are detected by a primary antibody against the viral antigen expressed by these cells. The primary antibody may be directly labeled or detected with a secondary labeled antibody.


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  • Many commercial laboratories will offer IFA tests for a variety of purposes.
  • 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 a 30-60 minute 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 Blood biochemistry: gamma globulin , but IgM detection is sometimes used in infectious disease serology) that is conjugated to a fluorochrome (typically fluoroscein isothiocyanate (FITC)).
  • The conditions of incubation of the secondary reagent vary with the particular assay.
  • Following this incubation, the slide is washed to remove unbound secondary antibody then mounted under an aqueous mountant.
  • The slide is then viewed by fluorescence microscopy at an appropriate wavelength to permit emission of fluorescence by the fluorochrome.
  • Similarly, the immunoperoxidase method has been adapted for use in such assays in which the fluorochrome-labeled secondary antibody is replaced by an enzyme-labeled secondary antibody.
  • Localization of this enzyme-labeled antibody is subsequently demonstrated by the use of a substrate-chromate and conventional light microscopy.



  • The IFA is an sensitive test that is often considered the gold standard of infectious disease serology.
  • Detection of FeLV antigen infected cells by immunofluorescence in blood smears is considered a reference technique for FeLV diagnosis equivalent to virus isolation.


  • The specificity of the test is limited by the specificity of the antibody present in serum.
  • In common with many serological tests, cross-reactive antibody cannot be distinguished in this assay, eg a dog may be seropositive when screened in an IFA to Ehrlichia canis Ehrlichia canis but may still have infection with Ehrlichia ewingii or Ehrlichia chaffeensis because there is serological cross-reactivity (shared antigenic epitopes) between the different species of Ehrlichia.
  • Cross-reaction of antibody between microbial genera can also occur, eg a dog infected with Toxoplasma may show positive serology to Babesiaas these organisms share common epitopes.
  • Such cross-reactions can be investigated by the use of PCR Polymerase chain reaction testing which can unequivocally identify the unique DNA sequences of respective infectious agents.
  • The pattern of the Immunofluorescence reaction observed helps to confirm the specificity of the result. For example, clear spirochaetes may be seen in leptospira IFA Canine leptospira antibody titer and a typical patter of infected cell fluorescence is seen in feline coronavirus and FIV IFA. For this reason IFA test are generally regarded as superior to ELISA and Immunochromatography techniques.

Predictive value

  • It should be remembered that serological tests indicate exposure to an infectious agent and do not necessarily imply current infection.
  • Seropositive animals may have eliminated an infection immunologically, or may have been successfully treated for the infection.
  • Detection of antigen, rather than antibody, is necessary to determine actual current infection.
  • Serology is particularly useful where exposure and clinical disease are consistent, eg FIV infection.
  • Serology can also be useful in diseases of a chronic or progressive nature where the original infecting agent may no longer be detectable, eg in cases of delayed presentation of acute viral infections with chronic sequelae.
  • The magnitude of the titer observed increases the predictive value of the positive test result for most serological tests.
  • For some serological tests using IFA higher titers are more commonly associated with clinical disease due to that pathogen.
  • Advice on result interpretation should always be sought from the laboratory performing the test.

Result Data

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Further Reading


Refereed papers

  • Recent references from VetMedResource and PubMed.
  • Burr P, Snodgrass D (2004) Demystifying diagnostic testing: serology. In Practice 26, 498-502.
  • Day M J (1996) IgG subclasses of canine anti-erythrocyte, anti-nuclear and anti-thyroglobulin autoantibodies. Res Vet Sci 61,129-135.
  • Iwasaki T, Shimizu M, Obata H et al (1996) Effect of substrate on indirect immunofluorescence test for canine pemphigus foliaceus. Vet Pathol 33, 332-336.

Other sources of information

  • Day M J (1999) Clinical Immunology of the Dog and Cat. Manson Publishing, UK.


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