3.1) is used primarily to detect and quantify antigen in a sample. This technique requires fewer steps than the others and is easier to perform since it only involves use of a single primary antibody labeled with a reporter enzyme. Substrate added to the sample interacts with the reporter enzyme to produce a product (e.g. color, fluorescence, or luminescence) that is measured with a plate reader. The direct method's use is limited to IHC, since it requires that the sample antigen is immobilized. The indirect technique (Figure 3.2) is the most popular type of assay and is used primarily to detect antibodies in a sample. This requires binding of antigen to the assay plate, adding the animal's serum (containing the antibody), and then adding detection antibody that is linked to a reporter enzyme along with substrate to be measured with the plate reader. The capture technique (Figure 3.3), sometimes called the “sandwich assay,” is a modified version that detects antigen in the sample and has superior sensitivity and specificity compared to the other techniques.
Figure 3.1 Direct ELISA. In the first stage the antigen (orange triangle) from the patient's serum is added to the plate where it is absorbed. This antigen can be a protein, hormone, etc. Next the labeled antibody (blue antibody) is added. The plate is washed between steps to remove unattached antibodies and antigens. Finally the substrate (yellow star) for the enzyme is added to produce a color change that can be identified and quantitated.
Figure 3.2 Indirect ELISA. The antigen (orange triangle) is adhered to the plate. The patient's serum with the patient's antibodies (yellow) is added. The labeled antibody (blue) is then added. The plates are washed between each step to remove unattached antibodies. Finally the substrate (yellow star) for the enzyme is added to produce a color change that can be identified and quantitated.
Other types of ELISAs exist, including a competitive ELISA for detecting very small antigens (haptens), an enzyme‐linked immunospot assay (ELISPOT) for quantifying proteins, and an in‐cell ELISA to detect proteins within living cells (Ji 2016).
ELISA tests are highly variable in their sensitivity and specificity, which is influenced by the use of polyclonal versus monoclonal detection antibodies, different plate types, washing techniques, reporter enzymes, incubation times, and much more. In veterinary dermatology, ELISA tests are used to measure antibodies to various infectious organisms (e.g. Sarcoptes sp., Blastomyces spp., and numerous tick‐borne parasites). ELISA technology is also the primary modality used for measuring serum levels of allergen‐specific IgE (Diesel and Deboer 2011; Lee et al. 2009, 2012, 2015; Plant et al. 2014). Intradermal testing is still considered the gold standard for identification of relevant allergens for inclusion in allergen‐specific immunotherapy, due to its ability to demonstrate an immunologic response (wheal and flare) to the injected allergen in the skin, the target organ in our veterinary species. However, ELISA testing does have some advantages. Notably, ELISA technology makes allergy testing possible for pets without access to a veterinary dermatologist, pets unable to stop medications such as steroids that interfere with intradermal testing, and pets with comorbidities that prohibit sedation required for intradermal testing. The first commercially available ELISA tests for allergen‐specific IgE used polyclonal antibodies and suffered from a low specificity. More specialized ELISA technology has since been developed. Heska Allercept® testing uses a recombinant form of the human mast cell high‐affinity receptor for IgE (Fc€RIα) for improved specificity to allergen‐specific IgE. Greer’s Aller‐g‐complete® ELISA test uses monoclonal antibody cocktails recognizing multiple epitopes on IgE for improved sensitivity, and it is currently investigating the use of inhibitor antibodies to prevent nonspecific IgE binding to cross‐reactive carbohydrate modalities on the surface of plant and insect antigens (Gedon et al. 2019; Levy and Deboer 2018; Piccione and Deboer 2019).
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
