Immunofluorescent antibody tests

Overview

• 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.

Uses

Other points

• Immunofluorescence antibody tests (IFA or IFAT) are widely used as a means of detecting seropositivity to an infectious agent. For example, serum antibodies to pathogens such as Feline Coronavirus, FIV, Toxoplasma, Neospora, Leptospira, Leishmania, Ehrlichia and Babesia.
• Other methods to detect antibodies in serum include neutralization tests, ELISA and immunochromatography.
• Indirect immunofluorescence is also used to detect serum antibodies to a range of autoantigens.
• The most common such applications in veterinary medicine are the detection of serum antinuclear antibody (ANA) or serum autoantibodies specific for epidermal autoantigens in the pemphigus diseases .

Result data

Normal (reference) values

• Indirect immunofluorescent assays will be reported as a titer of serum antibody.
• The titer is the inverse of the last dilution of serum that gave an unequivocally positive result in the assay, eg a final positive serum dilution of 1/128 equates to a titer of 128.
• The testing laboratory should provide an indication of the significance of the titer obtained.
• Many clinically normal animals, or animals with chronic inflammatory, infectious or neoplastic disease will have low titers of autoantibodies (particularly the antinuclear antibody), so the laboratory should indicate whether a clinically significant (in terms of suspected autoimmune disease) titer is present.
• Similarly, the laboratory should indicate whether the antibody titer to an infectious agent is significant, and if paired serum samples have been submitted, should comment on the relative titers in the two samples.

Sources

Publications

Refereed papers

• Recent references from 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, London.