Immunodiagnosis techniques
Immunodiagnostics is a diagnostic methodology that uses an antigen-antibody reaction as their primary means of detection. The concept of using immunology as a diagnostic tool was introduced in 1960 as a test for serum insulin. A second test was developed in 1970 as a test for thyroxine in the 1970s. It is well-suited for the detection of even the smallest of amounts of chemical substances. Antibodies specific for a desired antigen can be conjugated with a radiolabel, fluorescent label, or color-forming enzyme and are used as a “probe” to detect it. Well known applications include pregnancy tests, immunoblotting, ELISA and immunohistochemical staining of microscope slides. The speed, accuracy and simplicity of such tests has led to the development of rapid techniques for the diagnosis of disease, microbes and even illegal drugs in vivo. Such testing is also used to distinguish compatible blood types. The Enzyme-Linked ImmunoSorbent Assay or ELISA and the Lateral-Flow test, also known as the dipstick or rapid test, currently are the two predominant formats in immunodiagnostics.
Some of the important points related to immunodiagnosis are as follows:
- The detection of antibodies in serum may be of assistance in the diagnosis of an infectious disease. Specific antibodies may also be used to identify the presence of an antigen.
- The most sensitive and specific tests are those that directly detect the antigen or antibody of interest. These are called primary binding tests. An example of a primary binding test is an enzyme-linked immunosorbent assay (ELISA).
- Secondary binding tests tend to be the easiest to perform but are less sensitive than primary binding tests. Examples include precipitation and agglutination tests.
- Tertiary tests directly measure protection. They are usually complex and so may not lend themselves to rapid testing. An example is a virus neutralization test.
- Serological tests are judged by the number of false-positive results they generate (their specificity) and by the number of false-negative test results they generate (their sensitivity).
- In general highly sensitive tests tend to have low specificity, and vice versa.
Reagents Used in Serological Tests
Serum
The most common source of antibodies is serum obtained from clotted blood. Serum may be stored frozen and tested when convenient. If necessary, the serum can be depleted of complement activity by heating to 56° C for 30 minutes.
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. 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.
Primary Binding Tests
Primary binding tests are performed by allowing antigen and antibody to combine and then measuring the immune complexes formed. In order to measure these reactions, one of the reactants must be chemically labeled. Radioisotopes, fluorescent dyes, colloidal metals, and enzymes have all been used as labels in these tests.
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. 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.
Immunofluorescence Assays
Fluorescent dyes are commonly employed as labels in primary binding tests, the most important being fluorescein isothiocyanate (FITC). FITC is a yellow compound that can be chemically linked to antibodies without affecting their reactivity. When radiated with invisible ultraviolet or blue light at 290 and 145 nm, FITC re-emits visible green light at 525 nm. This fluorescence can be readily seen under a fluorescent microscope. FITC-labeled antibodies are used in the direct and indirect fluorescent antibody tests.
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. 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. 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.
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 . 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.
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. 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.
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.
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. 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. 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!).