Figure 3.3 Sandwich ELISA. Monoclonal antibody (yellow) for the antigen that is being tested is attached to a plate. The patient's serum with possible antigen (orange triangle) is then added to the plate. Next enzyme‐labeled monoclonal antibodies (blue) are added. The plate is washed between each step to remove unattached antigen and antibody. Finally the substrate (yellow star) is added to produce the color change.

      For now, intradermal allergy testing (skin testing) remains the most reliable method for identifying allergens for use in immunotherapy. Clinicians must remember that neither skin testing nor serologic testing is used to make the diagnosis of atopy. These tests are used to identify indoor allergens such as house dust mite that might be avoided, or to identify allergens for inclusion in allergen‐specific immunotherapy.

      Immunohistochemistry

      IHC staining is an ELISA‐based technique commonly used in veterinary medicine to detect antigens within tissue, specifically formalin‐fixed tissue. IHC has many applications as a diagnostic modality, since it detects molecular markers and proteins in cells that identify cell types and determines where in the cell the antigen of interest exists. For example, a chromogenic assay using rabbit anti‐Pythium antibodies is used to detect Pythium sp. hyphae within formalin‐fixed tissue from dogs (Brown et al. 1988). Colorimetric (chromogenic) and fluorescent assays are most commonly used for visualizing the antigen–antibody interaction in the tissue. The detection antibodies used may be monoclonal or polyclonal as described earlier, which can influence the binding sensitivity and specificity of the assay.

      Immunofluorescence

      Like IHC staining, IF is an ELISA‐based technique that detects antigen or antibody in tissue samples using antibodies labeled with fluorescent dyes (fluorochromes). IF primarily uses fresh frozen tissue fixed in acetone or methanol; however, suspensions of cells, cell cultures, beads, and microarrays can also be used (Severo et al. 2018). Direct and indirect techniques exist where the fluorochrome is bound directly to the agent‐specific antibody (direct IF) or attached to an anti‐immunoglobulin that recognizes the agent‐specific antibody (indirect IF). For example, direct IF is used to detect desmocollin‐1 antibodies in the skin of dogs with pemphigus foliaceus, whereas indirect IF is used to detect desmocollin‐1 antibodies in the serum of dogs with pemphigus foliaceus (Bizikova et al. 2012; Olivry et al. 2006; Severo et al. 2018). IF testing is not widely used diagnostically in veterinary dermatology compared to its growing popularity in human medicine. Instead, its use remains most popular within the research field due to lack of accessibility, the need for specialized equipment, and the ability to make a reliable diagnosis via other means such as clinical evaluation, infectious disease testing, and dermatopathology. Recent evaluation of direct and indirect IF for supporting the diagnosis of pemphigus foliaceus in the dog showed a 75% agreement of direct IF and a 100% agreement of indirect IF with histopathology (Severo et al. 2018). When IF testing becomes more available and affordable, it may be a useful tool for the diagnosis of pemphigus foliaceus.

      Flow Cytometry

      The physical and chemical characteristics of individual cells and particles suspended in a fluid can be analyzed using flow cytometry. This methodology uses a laser illuminating beam that passes over the molecules within the liquid sample as they advance single file through the flow chamber (Pedreira et al. 2013). This technique allows for rapid, objective, and quantitative evaluation of cells, even when present in small numbers within the sample. Flow cytometry uses light scatter and absorption to measure individual cells and has the added ability to sort cells based on these characteristics into separate collection tubes for future experiments. Flow cytometry is a useful tool for the diagnosis and study of hematologic neoplasia in veterinary medicine (Meichner et al. 2020).

Secondary Binding Tests

      Secondary binding tests measure antigen–antibody interaction in vitro. These tests measure the secondary effect, or consequence, of the antigen–antibody interaction. Agglutination, precipitation, neutralization (of bacteria, viruses, toxins, etc.), and complement activation are all examples of secondary binding tests (Tizard 2013). They are not as sensitive as primary binding tests, but they are much easier and faster to perform. In general, these tests are not used often in veterinary dermatology.

Tertiary Binding Tests

      These are in vivo tests that measure the protective effect of antibodies within circulation and provide information about the significance of the immune response (Tizard 2013). These tests are much more complicated to perform and are not used often in veterinary dermatology.

Polymerase Chain Reaction

PCR is a molecular copying technique useful for detecting very small amounts of DNA in a sample. PCR makes millions to billions of copies of specific regions of DNA, referred to as amplicons, in order to amplify, quantify, and further study the molecule of interest (Waters and Shapter 2014). The basic steps of PCR involve denaturing the DNA of interest using heat, annealing oligonucleotide pairs (primers) to desired regions of the DNA, and making copies of this DNA template using DNA polymerase. Each time this reaction takes place, the DNA is doubled, so that at the end of 20 cycles there are more than one million copies of the template DNA. Once the DNA is amplified, additional laboratory techniques such as quantification of a molecule of interest, DNA sequencing, DNA cloning, and ELISA can be used to study the DNA further. PCR tests provide rapid