Immunoassay Formats

Immunoassays can be divided into two types, heterogeneous and homogeneous immunoassays. Heterogeneous immunoassays involve a solid phase (microwell, bead) and require washing steps to remove unbound antigens or antibodies. Heterogeneous immunoassays can have a competitive or noncompetitive format.

Homogeneous immunoassays consist only of a liquid phase and do not require washing steps. Homogeneous immunoassays are faster and easier to automate than heterogeneous immunoassays. In addition, homogeneous immunoassays have competitive formats.

Types of Labels

The principles and applications of enzyme immunoassays, chemiluminescence, and fluorescent substances as labels are presented in this chapter (Table 12-1).

The original technique of using antigen-coated cells or particles in agglutination techniques may be considered as the earliest method for labeling components in immunoassays. Ideal characteristics of a label include the quality of being measurable by several methods, including visual inspection. The properties of a label used in an immunoassay determine how detection is possible. For example, coated latex particles can be detected by various methods—visual inspection, light scattering (nephelometry), and particle counting. The conversion of a colorless substrate into a colored product in enzyme immunoassay allows for two methods of detection, colorimetry and visual inspection.

Yalow and Berson developed the radioimmunoassay (RIA) method in 1959 using a radioactive label that could identify an immunocomponent at very low concentrations. In the 1960s, researchers began to search for a substitute for the successful RIA method because of the inherent drawbacks of using radioactive isotopes as labels (e.g., radioactive waste, short shelf life). Currently, chemiluminescent reactions have replaced most RIAs in the clinical laboratory. Relatively simple and cost-effective, chemiluminescence technology has sensitivity at least as good as that of an RIA.

Enzyme Immunoassay

There are two general approaches to diagnosing condition, diseases or conditions by immunoassay, testing for specific antigens or for antigen-specific antibodies. The enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA), is designed to detect antigens or antibodies by producing an enzyme-triggered color change.

The EIA method uses a nonisotopic label that offers the advantage of safety. EIA is usually an objective measurement that provides numerical results. Some EIA procedures provide diagnostic information and measure immune status (e.g., detect total antibody IgM or IgG).

The EIA method uses the catalytic properties of enzymes to detect and quantitate immunologic reactions. An enzyme-labeled antibody or enzyme-labeled antigen conjugate is used in immunologic assays (Box 12-1). The enzyme, with its substrate, detects the presence and quantity of antigen or antibody in a patient specimen. In some tissues, an enzyme-labeled antibody can identify antigenic locations.

Various enzymes are used in enzyme immunoassay (Table 12-2). Common enzyme labels are horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, and beta-galactosidase. To be used in an EIA, an enzyme must fulfill the following criteria:

In a representative EIA test, a plastic bead or plastic plate is coated with antigen (e.g., virus; Fig. 12-1). The antigen reacts with antibody in the patient’s serum. The bead or plate is then incubated with an enzyme-labeled antibody conjugate. If antibody is present, the conjugate reacts with the antigen-antibody complex on the bead or plate. The enzyme activity is measured spectrophotometrically after the addition of the specific chromogenic substrate. For example, peroxidase cleaves its substrate, o-dianisidine, causing a color change. In some cases, the test can be read subjectively.

The results of a typical test are calculated by comparing the spectrophotometric reading of the patient’s serum to that of a control or reference serum. The advantage of an objective enzyme test is that results are not dependent on a technician’s interpretations. In general, the EIA procedure is faster and requires less laboratory work than comparable methods.

Chemiluminescence

Chemiluminescence