Immunoassays

Chapter 2


Immunoassays





Introduction


Serology refers to the measurement of antigen-antibody interactions for diagnostic purposes, but is most often used to refer to the detection of antibodies in serum. In general, a blood sample is seroreactive (or seropositive) if it contains antibodies to a particular pathogen. Assays have been designed that detect different classes of antibodies in serum, most commonly IgG or IgM. Methods used to detect antibodies can also be applied to detect antigen. They can also be used on body fluids other than serum, such as urine, cerebrospinal fluid, and aqueous humor. Assays that measure antigen-antibody interactions for diagnostic purposes are most broadly referred to as immunoassays. Types of immunoassays include immunofluorescent antibody (IFA) tests, ELISAs, agglutination tests, gel immunodiffusion tests (AGID or gel ID tests), and Western blotting. Many of these assays involve the use of polyclonal antibodies, or, more commonly, monoclonal antibodies, which are commercially available. Polyclonal antibodies are produced in animals (e.g., rabbits or horses) as a normal antibody response to antigen exposure. Monoclonal antibodies are produced in almost unlimited amounts in tissue culture, and have a higher degree of antigenic specificity.


Some immunoassays, such as ELISA and IFA, provide quantitative results, expressed as optical density units (ODs) or titers (e.g., 1:800, or reciprocal titer = 800). Exposure to an infectious agent results in a rise in antibody titer over time (Figure 2-1). Usually, the first antibody class to be produced by plasma cells is IgM, after which IgG synthesis by plasma cells occurs. When exposure to a pathogen occurs for the first time, there is a lag phase of 5 to 7 days before IgM antibodies can be detected in the blood (primary antibody response). The antibody titer increases, and if the infectious agent is cleared, there is a progressive decline in the titer over weeks to months. The speed and magnitude by which the antibody titer rises depends on host factors and the infectious agent involved. For some diseases, such as leptospirosis, a significant rise occurs within 3 to 4 days of the onset of clinical signs. In secondary, or anamnestic, immune responses, the lag phase is shorter, the antibody titer increases more rapidly and to a greater magnitude, and antibodies may persist for longer periods of time (months to years).



Serologic diagnosis of infectious diseases relies on an understanding of the timing of the antibody response in relation to development of clinical signs for different infectious diseases. The results of serologic testing are interpreted differently for acute infections than they are for chronic or persistent infections. For infections with a short incubation period, such as leptospirosis or Rocky Mountain spotted fever, antibody titers are often negative in the first week of illness. In general, an increase in antibody titers over a 2- to 4-week period then occurs. Seroconversion is consistent with recent infection, and at least a fourfold increase in titer is required for the change to be considered significant. Because serologic test results can differ between laboratories that perform the same assay, the same laboratory should be used to determine the acute and the convalescent titer. Ideally, to minimize interassay variation, an aliquot of the acute specimen should be stored, frozen, and assayed at the same time as the convalescent specimen, although in reality this is often not done. A fourfold decline in antibody titer may also be consistent with recent infection, depending on the stage of illness at which serologic testing is performed. For chronic, persistent infections, such as FIV infection or canine leishmaniosis, a single, positive antibody titer can indicate active infection. Titer increases do not occur when infection persists beyond 1 to 2 months, and so acute- and convalescent-phase serology is not useful for diagnosis of chronic infections.




Diagnostic Methods



Immunofluorescent and Immunoperoxidase Antibody Assays


Direct IFA (also known as direct FA) detects antigen in clinical specimens. IFA involves the use of an antibody that has been tagged (conjugated) with a fluorescent label (such as fluorescein isothiocyanate) to detect the presence a specific antigen. IFA is performed directly on a glass slide, and fluorescence is detected by microscopy. The sensitivity of direct IFA is too low to detect individual virus particles or soluble antigen, so usually IFA detects antigen present in association with eukaryotic or bacterial cells. If the antigen is intracellular, pretreatment of the slide to permeabilize the cells may be necessary. The glass slide is then incubated with a solution that contains the fluorescent antibody, then washed to remove unbound antibody, and the cells are examined using a fluorescence microscope (Figure 2-2). Examples of the use of direct IFA in veterinary medicine include detection of Giardia oocysts, FeLV within monocytes in peripheral blood or bone marrow, or canine distemper virus within epithelial cells from a conjunctival scraping. Nonspecific fluorescence can result in false positives using these methods, but this can be overcome with the inclusion of proper controls and technical expertise. An alternative to the use of a fluorescein-conjugated antibody is the use of an antibody that has been conjugated to an enzyme such as horseradish peroxidase (immunoperoxidase). A substrate, most commonly 3,3′-diaminobenzidine (DAB), is then added to the slides. In the presence of hydrogen peroxide, DAB is converted to an insoluble brown precipitate, which can be visualized using conventional light microscopy. This technique is known as immunocytochemistry. The same method, when applied to tissue sections, is known as immunohistochemistry (IHC) (Figure 2-3).




Indirect IFA (also known as IFA testing, or IFAT) is generally used to establish a serum antibody titer to an infectious agent, but it can also be used to detect antigen. When used to detect antigen, it is more sensitive than direct IFA. For measurement of antibody titers, patient serum is serially diluted in the laboratory and reacted with a known antigen that has been fixed to glass slides. IFA slides are commercially manufactured for this purpose. Slides are then washed to remove unbound antibody. A secondary antibody, which is designed to react with the bound patient antibodies (e.g., dog IgG or dog IgM), is applied to the slides, which are then washed again. This secondary antibody has been conjugated to a fluorescent label. The slides are then examined using a fluorescence microscope. The highest dilution of serum that results in specific fluorescence is reported to the clinician as the antibody titer to the infectious agent of interest. Examples of the use of indirect IFA for serologic testing in dogs and cats include quantitative serology for some tick-borne infectious diseases (e.g., Ehrlichia canis, Anaplasma spp.).


More recently, veterinary immunofluorescence assays have been marketed that allow multiple antigen (or antibody) targets to be mounted on novel platforms, such as fluorescent beads or wells on the surface of a spinning silicon disc that resembles a compact disc. Automated silicon disc–based assays allow rapid and simultaneous analysis of hundreds of specimens, with dramatic reduction in labor associated with traditional IFA or ELISA assays, and reduced chance of false-positive or -negative assay results due to human error. A bead-based assay is currently available in the United States for detection of antibody to Borrelia burgdorferi (see Chapter 51). A silicon disc–based assay is available in the United States for detection of antibodies to B. burgdorferi, E. canis, and Anaplasma phagocytophilum and antigen to Dirofilaria immitis (Accuplex 4, Antech Diagnostics, Irvine, Calif). The performance of these assays in the field is under investigation.

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Jul 10, 2016 | Posted by in INTERNAL MEDICINE | Comments Off on Immunoassays
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