improves our understanding of mechanisms of resistance and how selection pressure drives microbial adaptability within the environment. This information can assist us when choosing therapies to target these abnormalities, or when avoiding therapies neutralized by such mutations. Furthermore, new drug development targeting these genetic mutations is paving the way for individualized and safe disease treatments in the future.
Understanding the genomic variations unique to specific diseases will influence the use of animal models of disease and will contribute to the One Health model of disease recognition and treatment. Rapid and sensitive diagnostic methodology can detect emerging diseases within a population, and track prevalence of diseases over time. Molecular diagnostics are very sensitive techniques for achieving a diagnosis when only a small amount of sample is available; however, the techniques are more complicated and more expensive compared to traditional dermatologic diagnostics. Additional limitations to consider include understanding that the presence of DNA does not prove an organism's viability, and measurement of titers does not indicate active infection. This chapter will focus on the updated diagnostic testing modalities available in veterinary dermatology, particularly molecular diagnostics.
Molecular Diagnostic Tests: How Do They Work and What Is Available?
Not every molecular diagnostic test is created equal. Each test uses a different technique to study a disease or immunologic response, and the data obtained from each test answers different questions. Some tests detect antigens while others detect antibodies. Some tests sequence fragments of DNA or entire genomes to detect the presence of an organism or genetic modification. Refer to Table 3.1 for a list of some of the dermatologic diagnostic tests available today. Also, not every laboratory is created equally. It is important to use reputable laboratories and if possible to be sure the tests they are offering have been verified.
Serology
Serologic assays detect antigens or antibodies in bodily fluids, particularly serum. Measurement of the immune complexes formed when antibody molecules bind reversibly to antigens is the basis of serology. Antigen tests used in the diagnosis of infectious diseases allow for early detection of organisms. Interestingly, serologic assays are used to measure any “antigen” of interest, including cytokines, white blood cells, proteins, and cellular by‐products. In contrast to antigen tests, antibody tests demonstrate the immunologic response to certain antigens by measuring serum antibodies against specific antigens not present or easily isolated from the tissue. There are three major techniques used for serologic assays: primary binding, secondary binding, and tertiary binding (Tizard 2013).
Primary Binding Tests
Primary binding tests directly demonstrate and measure antigen–antibody binding reactions using chemical labeling with radioisotopes, fluorescent dyes, or enzymes. These tests are the most sensitive technique for measuring detectable antigen or antibody in a sample. Examples of primary binding serologic assays include radioimmunoassays (RIA), immunofluorescence (IF) assays, ELISAs, immunohistochemistry (IHC), Western blotting, and flow cytometry.
Radioimmunoassays
RIA provides excellent sensitivity with a small sample size. Limitations of RIA include the specialized equipment needed, expense, the special handling and disposal of the radioactive isotopes used, along with decreased specificity. RIA detects minute amounts of antigen in a sample by using the concept of competitive binding, in which unlabeled antigen displaces radiolabeled antigen within an immune complex. In other words, as the concentration of unlabeled antigen from the patient increases, more of it binds to the antibody and displaces the labeled antigen. Then the amount of free labeled antigen is measured.
Different RIAs exist for the detection of both antigens and antibodies. These assays are also excellent for detecting small quantities of drugs and hormones in the blood. Some of the original serologic allergy tests created were radioallergosorbent tests (RAST) (Dolen 2001). RAST measure allergen‐specific immunoglobulin (Ig) E in the serum of atopic animals via a two‐step method. The animal's serum is first applied to antigen‐impregnated cellulose disks, then the disks are washed with radiolabeled anti‐IgE solution. IgE is quantified by the degree of radiolabeling measured. Over time, allergy testing in dogs and cats transitioned to ELISA modalities for the measurement of allergen‐specific IgE to avoid the use radioactive compounds.
Enzyme‐Linked Immunosorbent Assay
ELISA is a plate‐based assay that allows detection and quantification of antigens such as hormones, antibodies, enzymes, proteins, and peptides. This test is used frequently in veterinary medicine due to its diagnostic accuracy, affordability, accessibility, rapid turnaround time, and ability to be modified into a SNAPR assay for in‐clinic use. Detection strategies used include IF, chemiluminescence, and colorimetric (chromogenic) techniques.
Table 3.1 Common diagnostic tests available in veterinary dermatology. This table includes some of the common diagnostic testing modalities for dermatologic conditions, including hypersensitivities, infectious diseases, and genetic abnormalities. This table is not inclusive of every possible diagnostic test available, and the authors recommend that readers contact commercial and research laboratories for a more complete list of available diagnostic tests. Many research and university laboratories have the ability to develop new molecular assays, and represent an invaluable resource, especially when newly describing uncommon dermatologic disorders.
| Serologic allergy testing | |||||
|---|---|---|---|---|---|
| Test | Company | Type of test | Sample required | Information obtained | Website |
| SPOT Platinum+ | ACTT® and Spectrum Veterinary | ELISA | Serum | Quantitative assay to measure allergen‐specific IgE | www.acttallergy.com |
| Allercept | Heska | ELISA using high‐affinity receptor (FcꞓRIα) for IgE, which prevents non‐IgE antibody binding | Serum | Quantitative assay to measure allergen‐specific IgE. 86–92% agreement with Greer® Aller‐g‐complete | www.heska.com |
| Aller‐g‐detect™ Allergen Preliminary Panel | IDEXX | ELISA | Serum | Qualitative assay that tests for presence of environmental allergen‐specific IgE. If positive, follow up with quantitative analysis using Greer Aller‐g‐complete | www.idexx.com |
| Greer Aller‐g‐ complete® | IDEXX | Non‐competitive solid‐phase ELISA using macELISA technology (cocktail of biotinylated canine monoclonal antibodies specific to Fc region of IgE) | Serum |
Quantitative assay to measure allergen‐specific IgE with Heska Allercept
|
