Enzyme Immunoassays

Enzyme immunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA), systems were first developed during the 1960s. The basic test consists of antibodies bonded to enzymes; the enzymes remain able to catalyze a reaction yielding a visually discernible end product while attached to the antibodies. Furthermore, the antibody-binding sites remain free to react with their specific antigen. The use of enzymes as labels has several advantages. First, the enzyme itself is not changed during activity; it can catalyze the reaction of many substrate molecules, greatly amplifying the reaction and enhancing detection. Second, enzyme-conjugated antibodies are stable and can be stored for a relatively long time. Third, the formation of a colored end product allows direct observation of the reaction or automated spectrophotometric reading.

The use of monoclonal antibodies has helped increase the specificity of currently available ELISA systems. New ELISA systems are continually being developed for detection of etiologic agents or their products. In some instances, such as detection of RSV, HIV, and certain adenoviruses, ELISA systems may even be more sensitive than current culture methods.

Solid-Phase Immunoassay

Most ELISA systems developed to detect infectious agents consist of antibody directed against the agent in question firmly fixed to a solid matrix, either the inside of the wells of a microdilution tray or the outside of a spherical plastic or metal bead or some other solid matrix (Figure 9-12). Such systems are called solidphase immunosorbent assays (SPIA). If antigen is present in the fluid to be tested, stable antigen-antibody complexes form when the fluid is added to the matrix. Unbound antigen is thoroughly removed by washing, and a second antibody against the antigen being sought is then added to the system. This antibody has been complexed to an enzyme such as alkaline phosphatase or horseradish peroxidase. If the antigen is present on the solid matrix, it binds the second antibody, forming a sandwich with antigen in the middle. After washing has removed unbound.

labeled antibody, the addition and hydrolysis of the enzyme substrate causes the color change and completes the reaction. The visually detectable end point appears wherever the enzyme is present (Figure 9-13). Because of the expanding nature of the reaction, even minute amounts of antigen (>1 ng/mL) can be detected. The system just described requires a specific enzyme-labeled antibody for each antigen tested. However, it is simpler to use an indirect assay in which a second, unlabeled antibody is used to bind to the antigen-antibody complex on the matrix. A third antibody, labeled with enzyme and directed against the nonvariable Fc portion of the unlabeled second antibody, can then be used as the detection marker for many different antigen-antibody complexes (Figure 9-14). ELISA systems are important diagnostic tools for hepatitis B s (surface) and e antigens and HIV p24 protein, all indicators of early, active, acute infection.

A Specific antibody



Plastic well
^ Enzyme-conjugated

Enzyme substrate antibody

Enzyme substrate

Colored end product


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