• Enzyme-Linked Immunosorbent Assay (elisa)

The enzyme-linked immunosorbent assay (ELISA), also known as the enzyme immunoassay (EIA), is an immunologic test that uses an enzyme-mediated reaction as an indicator of antigen-antibody complex production. ELISA is an extremely sensitive technique for rapidly detecting specific antibodies and antigens. This method, which has many variations, depends on the conjugation of an enzyme to either an antigen or an antibody. The enzyme is detected by assaying for enzyme activity with its substrate. Virtually any enzyme that converts its substrate to a detectable product can be used as the indicator. Peroxidase and alkaline phosphatase are commonly utilized as enzymes while 5-aminosalicylic acid, orthophenylendiamine, and other substances are used as the substrate for peroxidase.

T here are two basic methods of EIA. The direct ELISA commonly detects antigens, and the indirect ELISA detects antibodies (Fig. 10). A microtiter plates with numerous shallow wells are used in both procedures. The test has become highly automated in many applications, such as testing for the AIDS antibodies. To measure antibody, known antigen is fixed on a solid phase (e.g., plastic microdilution plate), incubated with test material (e.g., patient’s serum dilutions), washed, and reincubated with an anti-immunoglobulin (antiglobulin) antiserum labeled with an enzyme (e.g., horseradish peroxidase). Enzyme activity, measured by adding the specific substrate and estimating the color reaction, is a direct function of the amount of antibody bound. The number of formed enzyme-antigen-antibody complexes corresponds to the intensity of substrate staining. Therefore, the activity of the enzyme can be determined by the intensity of post-incubation staining with the appropriate substrate by means of the automatic device. The results are registered by a spectrophotometer.

NOTE: For the mechanism and the procedure of ELISA see «PRACTICAL WORK».

• Radioimmunoassay (ria)

Standard antigen preparations labeled with radioactive iodine (¹²⁵ I) provide another highly sensitive method for detecting minute amounts of antigen. The common technique (liquid phase modification) of competitive radioimmunoassay (RIA) employs the following procedure:

1. A known antibody preparation is mixed with the sample for assay. Any specific antigen in this sample will complex with the antibody. This antibody contains no radioactive label, so these complexes will not be radioactive.

2. ¹²⁵I-labeled antigen is then added to this mixture to react with any remaining antibody that wasn’t bound by the antigen in the sample. The number of radioactive antigen-antibody complexes is thus inversely proportional to the amount of antigen in the sample. If the sample contains none of the antigen, for example, all the antibody will be available to combine with the radioactive antigen indicator.

3. All antigen- antibody complexes are precipitated and separated from solution, and the radioactivity of the precipitate is determined. (Unreacted ¹²⁵I antigen remains in solution and is discarded.) The radioactivity of precipitate is inversely proportional to the amount of antigen in the test specimen. If this sample had large amount of antigen, it would react with most of the antibody, leaving little or no immunoglobulin to react with labeled antigen; very few radioactive antigen-antibody complexes would form, and the precipitate would have little radioactivity. Conversely, if the original sample had small amounts of antigen, the binding sites of the antibodies would still be available to react with the labeled antigen, yielding a highly radioactive precipitate.

This variant of RIA is based on the competition for specific antibody between the labeled (known) and the unlabeled (unknown) concentration of material (e.g., antigen). Therefore, this method is termed competitive RIA. The concentration of the unknown (unlabeled) antigen or hapten is determined by comparing the results with those obtained using several concentrations of a predetermined standard antigen.

In the competitive RIA in solid phase modification specific antibodies to the tested antigen are sorbed on the surface of shallow wells of commercial microtiter plates. Then, the antigen-containing material to be tested is placed into the wells and incubated. If the antigen reacts specifically with the antibody adsorbed to the well, the antigen will be retained there when the well is washed free of unbound antigen. The ¹²⁵I-labeled antigen is then added. If the material contained the antigen specific to immobilized antibodies, some of the active centers would be engaged. In this case the labeled antigen could react only with remaining antibody that wasn’t bound by the antigen in the sample. The number of radioactive antigen-antibody complexes is thus inversely proportional to the amount of antigen in the known sample.

The indirect RIA can be also employed for detecting both “unknown” antibodies (serological diagnosis) and unknown antigens. In both cases an anti-globulin labeled antisera are used. To carry out the serological diagnosis by indirect RIA, the known antigen is adsorbed to the well surface and then the patient’s serum is added. If it contains specific antibodies, the antigen-antibody complex is formed on the well surface. After washing the anti-globulin antiserum labeled by ¹²⁵I is inoculated into the wells. The greater the concentration of antibodies to the antigen in the patient’s serum, the larger the level of the radioactive label linked to the well surface.

RIA is highly sensitive method applied to the assay of hormones or drugs in serum. A specialized RIA, the radioallergosorbent test (RAST), is used to measure the amount of serum Ig E antibody that reacts with a known allergen (antigen).