ISSN 2398-2950      

Indirect immunofluorescence


Michael Day

Helen Milner


  • The indirect immunofluorescence method is used to detect serum antibodies, most often antibodies specific for an infectious agent or an autoantigen.
  • Serum 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.


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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, but IgM detection is important 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-chromagen and conventional light microscopy.



  • The IFA is an sensitive test that is often considered the gold standard of infectious disease serology.


  • 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 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 Babesia gibsonias these organisms share common epitopes.
  • Such cross-reactions can be investigated by the use of PCR PCR (Polymerase chain reaction)  testing which can unequivocally identify the unique DNA sequences of respective infectious agents.

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.

Result Data

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Further Reading


Refereed papers

  • Recent references from VetMedResource and PubMed.
  • Harrus S, Alleman A R, Bark H, Mahan S M & Waner T (2002) Comparison of three ELISAs with the indirect immunofluorescent antibody test for the diagnosis of canine infection with Ehrlichia canisVet Microbiol 86, 361-368 PubMed.
  • Scalone A, De Luna R, Oliva G et al (2002) Evaluation of the Leishmania recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of the standardized ELISAVet Parasitol 104, 275-285 PubMed.
  • Day M J (1996) IgG subclasses of canine anti-erythrocyte, anti-nuclear and anti-thyroglobulin autoantibodiesRes Vet Sci 61,129-135 PubMed.
  • Iwasaki T, Shimizu M, Obata H et al (1996) Effect of substrate on indirect immunofluorescence test for canine pemphigus foliaceusVet Pathol 33, 332-336 PubMed.

Other sources of information

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


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