For both antigen and antibody assays, detection methods such as fluorescence, color-linked enzyme reactions, and radioisotope labeling can be used to detect antigen:antibody complexes with varying levels of sensitivity.
Immunofluorescence assays (IFAs) involve a fluorescently labeled antibody detected with a fluorescence microscope, which increases assay sensitivity. IFA testing can be performed on blood smears or tissue sections and allows localization of the source of the positive signal.2 IFAs are commonly used to detect antibodies to infectious agents, including rickettsial organisms and Babesia spp. ELISAs involve an antibody linked to an enzyme that changes color when exposed to a substrate. Some tests provide more information than others. For example, with feline leukemia, readily available ELISA technology only tests for viral antigen in serum, which can be free or cell-associated. With an IFA, the fluorescent signal can be localized to WBCs, thus helping identify bone marrow involvement and persistent infection.
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Radioisotopes are also used to label antibodies to provide more sensitive detection of low abundance targets.2 Examples of radioimmunoassays include tests for cortisol, thyroid hormones, and cyclosporine. Although radioimmunoassays have often been considered the gold standard for detecting low abundance targets such as drugs and hormones, use of other increasingly sensitive nonradioactive assays that avoid the hazards of radiation is increasing.3,4
Other immunologic methods to detect serum antibodies include the microscopic agglutination test, which is often used to detect antibodies to Leptospira spp; serum neutralization; gel immunodiffusion; and immunoblotting (or Western blotting).
The following immunoassay applications (ie, serology, immunohistochemistry, flow cytometry) can be used to determine titers for infectious disease testing, special stains for biopsy samples, and immunophenotyping.
FIGURE 2 Titer determination for serum antibodies. Various dilutions of serum are incubated on slides or in plates precoated with antigen. A detection antibody is then applied to recognize bound serum antibodies. The titer is the highest dilution of serum (ie, the most dilute serum sample) that still causes a positive test result.