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IFA tests can be either direct or indirect. Direct fluorescent antibody tests can detect the presence of specific antigens within a tissue sample. With IFA, antibody labeled with a fluorescent marker is applied to a tissue sample that has been treated with antibody specific to the antigen of interest. The fluorescent marker binds to the antibody, revealing the presence of antigen when examined under a dark‐field microscope with an ultraviolet light source. This method is commonly used for the detection of rabies virus in brain tissue and FeLV in white blood cells. Indirect fluorescent antibody tests can be used to detect the presence of either tissue antigen or, more commonly, serum antibody. This technique is similar to direct fluorescent antibody testing, however, the sample to be tested is treated with antibodies against the molecule of interest prior to fluorescent labeling. This allows for amplification of the fluorescent signaling, determination of specific classes of antibody in the sample (i.e. IgG, IgM, IgA, etc.), and a quantitative estimation of the amount of antibody in the sample (Tizard 2013). Indirect fluorescent antibody tests are commonly used for the detection of brucellosis and rickettsial organisms.

Western blotting is a method of antigen or antibody detection primarily used for complex proteins including microorganisms and parasites. With this technique, protein antigens are separated via electrophoresis and immobilized on a nitrocellulose membrane. The antigens are then visualized through the use of an enzyme or radioimmunoassay (Tizard 2013). Western blotting is commonly used for the detection of antibodies against FIV, Borrelia spp., Ehrlichia spp., Leishmania, and Neorickettsia spp.

Immunohistochemistry can be utilized to identify specific antigens within histopathological tissue specimens. Prepared tissue sections are treated with an enzyme‐labeled antibody that binds to the antigen of interest. When exposed to an enzyme substrate, the labeled complexes produce a distinct brown color which can be identified by the pathologist. Immunohistochemistry has a wide variety of applications but is most commonly used for the diagnosis of viral diseases such as feline infectious peritonitis, canine distemper virus, and canine parvovirus and is frequently performed on postmortem tissue samples.