Diagnostics immunology (Proceedings)

Immunology plays a huge role in diagnostics for infectious diseases. In addition, various assays are able to assess the immune system itself in an animal.

For infectious disease diagnostics, specific assays for pathogen detection utilize pathogen-specific antibody. There are a wide variety of assay methodologies that vary in sensitivity and specificity.

Pathogen-specific antibody is used to bind antigen in sample. Assays vary in sample type, how visualization of binding occurs, and the sensitivity and specificity. The pathogen-specific antibody may be labeled with a fluorescent tag, a radioisotope, or an enzyme (substrate addition leads to color change). Depending on the assay, samples can include tissue impressions/scrapings, pelleted cells, formalin-fixed tissue (IHC). The quality of specimen affects results. These assays generally have a fast turn-around time and are inexpensive. In addition, the orrganism does not have to remain viable. Sensitivity varies, and is generally intermediate to low; thus a positive result "rules in" but negative result may not rule out a pathogen. As these are specific assays, they will not reveal presence of other agents unless specifically targeted.

Identification of virus-specific antibody:

In these assays, antibody is the unknown rather than the antigen. It may or may not be quantified.

Methods can be divided into three categories.

     • Direct detection of antibody.

     • Detection of antibody-facilitated action.

     • Detection of antibody-facilitated inhibition.

Direct detection of antibody:

Binding of patient's antibody to capture antigen anchored on a slide or membrane.Antibody is detected through labeled anti-immunoglobulin (i.e. anti feline IgG). Examples are immunofluorescence assay, ELISA, and western blot. They vary in how the capture antigen is anchored and how the antigloublin is tagged. Some serologic assays quantitate the amount of antibody in the serum:

Detection of antibody-facilitated action:

Binding of patient's antibody to the antigen leads to an activity due to the formation of the immune complex. For example, complement fixation where binding leads to activation of complement cascade; agglutination or precipitation where the pathogen is clumped by antibody and precipitates out of solution such as agar gel immunodiffusion (AGID) assays.

Detection of antibody-facilitated inhibition:

Binding of the patient's antibody to the pathogen results in inhibition of an activity by the pathogen. Generally, these assays measure antibody to very specific surface epitopes. Hemagglutination inhibition takes advantage of the ability of some pathogens to agglutinate RBC's. Antibody binding inhibits the ability of a virus to agglutinate RBC's, and the RBC pellets out in the well. Serum Virus Neutralization assays are another example; in this assay, antibody binding inhibits the ability of a virus to infect and lyse cells in culture (antibody "neutralizes" the pathogen).

Criteria for diagnosing primary infection

     • Four-fold increase in antibody levels between acute and convalescent titers.

     • Seroconversion.

     • Presence of IgM.

If onset of clinical signs coincides with antibody production, active disease may be diagnosed.

For some pathogens, disease occurs before antibody production – serology would thus be retrospective.

Some pathogens cause disease months or years later, such as FIV; detection of antibodies is sufficient to diagnose infection. However, one can rarely determine the current infection status based on the magnitude of a single sample (exception would be FIV in unvaccinated cat). One can mimic paired by sampling portion of the population – animals acutely ill, and animals that are recovered. It is important to notify the lab when submitting a convalescent sample, as acute and convalescent must be run side by side for comparison to be valid. In addition, one cannot compare titers between labs!

So what does the titer mean? If negative: No recent exposure/infection; Early infection.

If positive: Has been exposed/infected sometime in the past – do not know when.

High titer (relative term, determined by testing laboratory) - correlates with but NOT confirmatory of active infection.Communication with the diagnostic laboratory used is critical to the proper interpretation of any test result.

Evaluation of the functionality of the animal's immune system is sometimes needed when immunosuppression or immunodeficiency are suspected. Compared to human medicine, there are few assays for use in veterinary medicine. The ability of lymphocytes to respond to stimulation and activation can be assessed through incubation with mitogenic substances. These substances may target multiple or specific subsets of lymphocytes. Functional complement deficiency can be assessed by the complement hemolytic assay, but is not available for routine diagnostics. Neutrophil phagocytic and bactericidal activity can be evaluated with a killing assay using bacteria coated with complement. Flow cytometry holds a great deal of promise for evaluating various parameters of the immune response, including levels of lymphocyte subsets.

Assays for immune mediated diseases also employ immunologic principles. The antinuclear antibody test (ANA) detects antibody reactive with nuclear components, while the Coombs test detects antibody or complement bound to the surface of RBC's.

References

Kania, S. K. 2010. Diagnostic Assays for Immunologic Diseases in Small Animals. VCNA, 40(3):469-471.

Kennedy, M. A. 2005. Methodology in Diagnostic Virology. VCEAP, 8(1):7-26.