Enzyme immunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA), was developed during the 1960s. The basic method 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, human immunodeficiency virus (HIV), and certain adenoviruses, ELISA systems may even be more sensitive than culture methods.

Solid-Phase Immunoassay

Most ELISA systems developed to detect infectious agents consist of antibody 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 1). Such systems are called solid phase immunosorbent assays (SPIA). If antigen is present in the specimen, stable antigen-antibody complexes form when the sample is added to the matrix. Unbound antigen is thoroughly removed by washing, and a second antibody against the antigen 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 2). Because of the expanding nature of the reaction, even minute amounts of antigen (greater than 1 ng/mL) can be detected. These systems require 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 3). ELISA systems are important diagnostic tools for hepatitis Bs (surface) and hepatitis Be (early) antigens and HIV p24 protein, all indicators of early, active, acute infection.

Fig1. ProSpecT Giardia/Cryptosporidium Microplate Assay. A, Breakaway microwell cupules and kit components. B, Positive (yellow)  changing to blue following the addition of the stop reagent. The negative reactions remain clear. (Courtesy Remel, Inc., Lenexa, Kan.)

Fig2.  Principle of direct solid-phase enzyme immunosorbent assay (SPIA). A, Solid phase is microtiter well. B, Solid phase is bead. 

Fig3. Principle of indirect solid-phase enzyme immunosorbent assay (SPIA).

Membrane-Bound SPIA

The flow-through and large surface area characteristics of nitrocellulose, nylon, and other membranes have been exploited to enhance the speed and sensitivity of ELISA reactions. An absorbent material below the membrane pulls the liquid reactants through the mem brane and helps to separate nonreacted components from the antigen-antibody complexes bound to the membrane; washing steps are also simplified. Membrane bound SPIA systems are available for several viruses (Figure4), group A beta-hemolytic streptococci antigen directly from throat swabs, and group B streptococcal antigen in vaginal secretions. In addition to their use in clinical laboratories, these assays are expected to become more prevalent for home testing systems.

Fig4. Directigen respiratory syncytial virus (RSV) membrane-bound cassette. A, Positive reaction. B, Negative reaction. (Courtesy  Becton Dickinson Diagnostic Systems, Sparks, Md.)

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المزيد

الآخبار الصحية

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المزيد

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مواضيع ذات صلة

Interpretation of Serologic Tests

Features of the Humoral Immune Response Useful in Diagnostic Testing

Immunofluorescent assays

Particle Agglutination

Precipitation Tests

Production of Antibodies for use in Laboratory Testing

Investigation of Strain Relatedness/Pulsed-Field Gel Electrophoresis

Detection of Antimicrobial Resistance

The Light Microscope

Identification of Microorganisms Grown in Culture

Direct Detection of Microorganisms

Laboratory Manifestations of Iron Overload