Karen F. Snowden Immunodiagnostic methods for a range of parasitologic infections are selectively available at fee‐for‐service diagnostic laboratories and as point‐of‐care tests that can be conducted in a clinical setting. There are two basic approaches in designing an immunologic test. Antigen detection tests identify specific parasite‐associated compounds in blood, serum or fecal suspensions that indicate the presence of the organism in the host. Alternatively, antibody detection tests show the host immune response to a parasite through the production of specific antibodies. In order to have a positive test result, it is assumed that the host animal is immunologically competent to react to the pathogen and that a sufficient time of exposure has occurred for the animal to produce detectable antibodies. There are a variety of test formats for immunodiagnostic tests. The enzyme‐linked immunosorbent assay (ELISA) is designed with a series of wells in a plate or tray with an end result indicated colorimetrically (Fig. 4.1). The lateral flow immunochromatographic assay uses similar principles and reagents in a cassette format, and works by capillary action with a series of reagents moving along a membrane with the end result indicated as a colored dot or line on the membrane (Fig. 4.2). Both of these test formats can be designed as antigen or as antibody detection assays, and both test formats have been developed for use with blood, serum, or feces. Most ELISA tests are designed for processing sample batches in a diagnostic lab setting. One benefit of this test format is that the intensity of color generated in the reaction is measurable using a spectrophotometer and is generally proportional to the antigen/antibody that is being detected. Therefore the ELISA may be used as a quantitative measure in a carefully calibrated test. The immunochromatographic tests are designed to give positive/negative results and are not generally designed to be quantitative; therefore the intensity of the colored dot/line is not necessarily proportional to the amount of antigen/antibody detected. Fig. 4.1 Schematic ELISA antigen detection procedures: (A) test surface: polystyrene well; (B) parasite‐specific capture antibody (may be monoclonal or polyclonal); (C) parasite antigen in serum of the animal patient; (D) detecting reagent parasite‐specific antibody labeled with an enzyme; (E) visualizing step: if enzyme is present, it acts on soluble substrate to produce color, which can be evaluated visually or measured spectrophotometrically. Fig. 4.2 Schematic of antigen detection using an immunochromatographic lateral flow test. Immunochromatographic tests and selected ELISA assays are available as point‐of‐care tests that can be performed in a clinical setting on one or several samples in a relatively rapid time frame. Two of the most widely used parasitologic immunochromatographic or ELISA tests in companion animal medicine are the heartworm antigen test, which detects antigens primarily produced by adult female Dirofilaria immitis and detected in blood, serum, or plasma, and the fecal antigen test for Giardia duodenalis. Since these types of assays are convenient, generally inexpensive, and relatively easy to perform in a clinical setting, it is likely that more point‐of‐care tests for additional parasites may become commercially available in the future. Fig. 4.3 Comparison of common antibody detection procedures: indirect fluorescent antibody (IFA, left) test and enzyme‐linked immunosorbent assay (ELISA, right). A, test surface: glass slide (IFA) or polystyrene well (ELISA); B, parasite antigen: whole parasite, such as cultured tachyzoites and promastigores (IFA), or soluble parasite antigen, which can be a crude homogenate, a purified protein, or a recombinant protein (ELISA); C, serum of the animal patient, which may contain parasite‐specific antibodies; D, detecting reagent for host‐specific antibody: host‐specific antibody labeled with a fluorochrome such as fluorescein (IFA) or host‐specific antibody labeled with an enzyme (ELISA); E, visualizing step: specific UV wavelight from microscope causes fluorescein (FITC) to emit yellow‐green fluorescence (IFA); F, visualizing step: if enzyme is present, it acts on soluble substrate to produce color, which can be evaluated visually or measured spectrophotometrically. Another commonly used format is the indirect immunofluorescent assay (IFA), which is an antibody detection test designed for use with serum or plasma (Fig. 4.3). These tests are routinely performed in the diagnostic lab setting because a compound microscope equipped with appropriate barrier filters and a UV light source is needed to conduct the test. The test result is typically expressed as a “titer” and the IgG antibody isotype is usually the immunoglobulin that is detected in the test. The titer value is the reciprocal of the highest dilution of serum/plasma where the test remains positive. Different test formats (ELISA vs. IFA most commonly) have different thresholds of antibody detection. Therefore, the positive/negative cutoff value for each standardized assay should be provided by the laboratory performing the test in order to adequately interpret the meaning of the antibody titer as positive/negative or high/low. For example, the cutoff value for an IFA test might be a 1 : 10 dilution, while a similar test in an ELISA format might have a 1 : 100 dilution cutoff value as positive.
CHAPTER 4
Immunodiagnostic and Molecular Diagnostic Tests in Veterinary Parasitology
IMMUNODIAGNOSTIC METHODS IN PARASITOLOGY
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