As in fluorescent antibody tests, the antibody in EIAs is conjugated with a marker that can
be detected when an antigen-antibody reaction has taken place. In EIAs, the marker is an
enzyme, typically alkaline phosphatase or horseradish peroxidase. These enzymes catalyze the
breakdown of a colorless substrate to a colored end-product. To visualize the binding of antigen
and the enzyme-linked antibody, the appropriate substrate for the enzyme must be
added. A positive reaction results in the production of a colored end-product that can be detected
visually or measured quantitatively in a spectrophotometer.
Two basic formats for EIA testing are in use. In the first, monoclonal antibody
specific for the antigen sought is bound to a solid surface such as plastic tubes, beads, or wells
of a microtiter tray. The clinical sample is added to this solid surface followed by incubation
and washing steps. If the antigen is present in the sample, it will bind to the antibody and
unbound material is washed away. Now, the enzyme-labeled antibody is added to detect the
antigen-antibody complex. This step is accomplished either in a direct
the direct method, the second antibody, which is conjugated to the enzyme, reacts with antigen
bound by the first antibody on the solid surface. In the indirect method, two additional
antibodies are needed to develop the reaction. The first is unlabeled antibody specific for the
bound antigen and the second is an enzyme-labeled antibody that reacts with the first antibody.
In this way, the indirect EIA is similar to the IFA test. Finally, in both test methods the
substrate for the enzyme is added. The amount of colored end-product that develops indicates
the amount of antigen present in the clinical sample. Figure 19.3 illustrates direct and
indirect EIA methods.
EIA kits for detecting certain parasites, rotavirus, and other enteric viruses, and
toxins of diarrhea pathogens, are available in this format. Colorplate 25
illustrates such a kit.