Antiglobulins

Immunoglobulins are antigenic when injected into an animal of a different species. For example, purified dog immunoglobulins can be injected into rabbits. The rabbits respond by making specific antibodies called antiglobulins. Depending on the purity of the injected immunoglobulin, it is possible to make nonspecific antiglobulins against immunoglobulins of all classes, or very specific antiglobulins directed against single classes. Antiglobulins are essential reagents in many immunological tests.

Monoclonal Antibodies

Hybridoma-derived monoclonal antibodies are pure and specific, can be used as standard chemical reagents, and can be obtained in almost unlimited amounts (Chapter 15). As a result, monoclonal antibodies frequently replace conventional antiserum as reagents in immunodiagnostic tests.

Specific Antibodies

When detecting antigens in tissues or body fluids, the first steps may involve the use of a specific antibody against the antigen of interest. Although these antibodies are often made by immunizing mammals, there is a growing interest in using chicken IgY antibodies. Birds may react very strongly against mammalian antigens. Chickens produce large amounts of IgY antibodies that become concentrated in the egg yolk. It may be much more convenient to harvest large amounts of antibody from egg yolks than have to bleed animals repeatedly. Antiglobulins may then be used to detect the bound IgY.

Radioimmunoassays for Antibody

The radioallergosorbent test (RAST) measures specific IgE in the serum of allergic animals. In this technique, antigen-impregnated cellulose disks are immersed in test serum so that any antibody binds to the antigen. After washing to remove unbound antibody, the disk is immersed in a solution containing radiolabeled antiglobulin (e.g., anti-IgE). The antiglobulin binds only if IgE has bound to the antigen. The amount of radioactivity bound to the disk is therefore a measure of the level of specific IgE antibody activity in the serum.

Radioimmunoassays for Antigen

Competitive immunoassays are based on the principle that unlabeled antigen will displace radiolabeled antigen from immune complexes (Figure 41-1). These tests are exquisitely sensitive and are commonly used to detect trace amounts of drugs. The antigen (or drug) is labeled with a radioactive isotope such as tritium (H³), carbon-14, or iodine-125. When radiolabeled antigen is mixed with its specific antibody, the two combine to form immune complexes that can be precipitated out of solution. Any radioactivity remaining in the supernatant fluid is due to the presence of unbound antigen. If unlabeled antigen is added to the mixture, before adding the antibody, it will compete with the radioactive antigen for antibody-binding sites. As a result, some labeled antigen will be unable to bind, and the amount of radioactivity in the supernatant will increase. If a standard curve is first constructed based on the use of known amounts of unlabeled antigen, the amount of antigen in a test sample may be measured by reference to this standard curve.

Direct Fluorescent Antibody Tests

Direct fluorescent antibody tests are used to identify the presence of antigen in a tissue sample. Antibody directed against a specific antigen such as a bacterium or virus is first labeled with FITC. A tissue section or smear containing the organism is fixed to a glass slide, incubated with the labeled antiserum, and then washed to remove any unbound antibody (Figure 41-2). When examined by darkfield illumination under a microscope with an ultraviolet light source, the organisms that bind the labeled antibody will fluoresce brightly. This test can identify the presence of small numbers of bacteria in a sample. For example, it can be used to detect M. avium subspecies paratuberculosis in feces, or to detect bacteria such as Dichelobacter nodosus, Listeria monocytogenes, or clostridia in diseased tissues (Figure 41-3). It may also be employed to detect viruses in tissue culture or in tissues from infected animals. Examples include the detection of rabies virus in the brains of infected animals or feline leukemia virus in infected cat leukocytes (Figure 38-3).

Indirect Fluorescent Antibody Tests

Indirect fluorescent antibody tests can be used to measure antibodies in serum or to identify specific antigens in tissues or cell cultures. When measuring antibody levels, antigen is employed as a tissue smear, section, or cell culture on a slide or coverslip. This is incubated in serum suspected of containing antibodies to that antigen. The serum is then washed off, leaving only specific antibodies bound to the antigen (Figure 41-4). These bound antibodies may then be visualized by incubating the smear in FITC-labeled antiglobulin. When the unbound labeled antiglobulin is removed by washing and the slide examined, the presence of fluorescence indicates that antibody was present in the test serum. The quantity of antibody in the test serum may be estimated by examining increasing dilutions of serum on different antigen preparations.

The indirect fluorescent antibody test has two advantages over the direct technique. Since several labeled antiglobulin molecules will bind to each antibody molecule, the fluorescence will be considerably brighter than in the direct test. Similarly, by using antiglobulins specific for each immunoglobulin class, the class of the specific antibody may also be determined.

Particle Concentration Fluorescence Immunoassays

Immunofluorescence assays can be automated and quantitated by means of particle immunoassays (Figure 41-5). For example, antigen-coated, submicrometer polystyrene particles can be mixed with test serum. After incubation, the particles are recovered by vacuum filtration, washed to remove unbound antibody, and exposed to a fluorescent antiglobulin. After filtering the suspension again and washing to remove unbound antiglobulin, the particle suspension can be placed in a spectrofluorometer and the intensity of particle-bound fluorescence measured. This provides a measure of the level of antibodies in the test serum. A very useful variation on this is the competitive assay used as a rapid test for antibodies to Brucella abortus in cattle. In this case, Brucella antigen-coated polystyrene particles are mixed with a standard amount of fluorescent anti-Brucella serum and the serum under test. If positive, the unlabeled test serum blocks the binding of fluorescent antibodies to the particles. The more antibody in the test serum, the greater is the inhibition of fluorescent antibody binding.

Microwell Enzyme-Linked Immunosorbent Assay Tests

The most common form of ELISA is used to detect and measure specific antibodies. In order to perform this assay, microwells in polystyrene plates are first filled with an antigen solution (Figure 41-6). Proteins bind firmly to polystyrene surfaces, so that after unbound antigen is removed by vigorous washing, the wells remain coated with a layer of antigen. These coated plates can be stored until required. The serum under test is added to the wells. Any antibodies in the serum will bind to the antigen layer. After incubation and washing to remove unbound antibody, the presence of any bound antibodies can be detected by adding a solution containing an antiglobulin chemically linked to an enzyme. This labeled antiglobulin binds to the antibody and, following incubation and washing, can be detected and measured by adding a solution containing the enzyme substrate. The enzyme and substrate have been selected to ensure that a colored product develops in the tube. The intensity of the color that develops is therefore proportional to the amount of enzyme-linked antiglobulin that is bound, which in turn is proportional to the amount of antibody present in the serum under test. The color intensity may be estimated visually or, preferably, by spectrophotometry.

One modification of this technique is the antibody sandwich ELISA, which can be used to detect and measure a specific antigen (Figure 41-7). The wells in polystyrene plates are coated with specific antibody (capture antibody) before testing. To conduct the test, the antigen solution to be tested is added to each well. The capture antibody will bind any antigen present in the test solution. This step is followed, after washing, by specific antibody, which also binds the antigen (the detection antibody). After washing to remove unbound antibody, enzyme-labeled antiglobulin, and substrate, as described for the indirect technique, are added. (It is important that the capture antibody and the detection antibody are from a different species and that a species-specific antiglobulin is used for visualization of the detection antibody. This will avoid false-positive results caused by binding of the antiglobulin to the capture antibody in the absence of antigen.) In this assay, the intensity of the color reaction is related directly to the amount of bound antigen. Because these tests involve the formation of antibody-antigen-antibody layers, they are called sandwich ELISAs. Sandwich ELISAs are used to detect circulating virus in blood from cats with feline leukemia.

Another common modification of this technique is the labeled-antigen ELISA used to detect antibodies. This is favored in manufactured diagnostic kits. The antigen is bound to the microwells before testing (Figure 41-8). The serum to be tested is added, followed, after washing, by labeled antigen. Any serum antibodies present will bind the labeled antigen to the microwell where it can be measured.

A competitive ELISA can be used to measure hapten molecules or viral antigens (Figure 41-9). In this technique, the microwell is coated with specific antibody before testing. In a single reaction, the test sample and enzyme-labeled antigen are placed in the well where the antigens compete for the antibody-binding sites. The amount of labeled antigen bound to the microwell is inversely related to the concentration of antigen in the test sample. This technique is faster than other ELISA techniques. It can be made very sensitive if the sample antigen is permitted to react with the antibody before the labeled antigen is added.

Western Blotting

One solution to the problem of identifying protein antigens in a complex mixture is by use of a technique called Western blotting. This is a three-stage primary binding test (Figure 41-10). Stage 1 involves electrophoresis of a protein mixture on gels so that each component is resolved into a single band. Stage 2 involves blotting or transfer of these protein bands to an immobilizing nitrocellulose membrane. This is accomplished by placing the membrane on top of the gel and sandwiching the two between sponges saturated with buffer. The membrane-gel sandwich is supported between rigid plastic sheets and placed in a buffer reservoir, and an electrical current is passed between the sponges. The protein bands are transferred from the gel to the membrane without loss of resolution.

The third stage involves visualization of transferred antigens by means of an enzyme immunoassay or radioimmunoassay. When an enzyme immunoassay is employed, the membrane is first incubated in specific antiserum. After the membrane has been washed, an enzyme-labeled antiglobulin solution is added. When this is removed by washing, substrate is added, and a color develops in the bands where the antibody has bound to antigen. When isotope-labeled antiglobulin is used, an autoradiograph must be made and the labeled band identified by darkening of a photographic emulsion. Western blotting is used to identify the important antigens in complex microorganisms or parasites (Figure 41-11). A variant form of the Western blot is the dot blot. Antigen solution is drawn through a nitrocellulose membrane so that any protein binds to the membrane. The presence of the antigen can be determined using specific antiserum and enzyme-labeled antiglobulin in sequence. After exposure to enzyme substrate, the presence of a stained dot is a positive reaction. (Use of nasal washings as a source of the antigen, such as when trying to detect respiratory viruses, is called a snot-blot!).