Enzyme immunoassay (EIA) to detect antigens

This short animation demonstrates detection of specific antigens using the enzyme immunoassay. This resource was developed by Cary Engleberg of the University of Michigan. It is part of a larger learning module about laboratory methods for clinical microbiology. The full learning module, editable animation, and video transcript are available at http://open.umich.edu/education/med/oernetwork/med/microbiology/clinical-microbio-lab/2009. Copyright 2009-2010, Cary Engleberg. This is licensed under a Creative Commons Attribution Noncommercial 3.0 License http://creativecommons.org/licenses/by-nc/3.0/.

(English captions by Andrea Matsumoto, University of Michigan.) Many simple rapid diagnostic tests detect
specific antigens in biological samples by

using an enzyme immunoassay.

The purpose of this animation is to explain
how a prototype of this assay works.

The enzyme immunoassay can be done in a multi-well
microtiter plate or on any other solid adherence

surface.

I will use the microtiter plate in this example
so, lets take a closer look at one of the wells

of this assay to see what happens during the
performance of the assay.

The plate is prepared to perform a particular
assay by coating the wells with antibodies

that bind to the antigen of interest.

Then the wells are filled with the clinical
sample, which could be a sample of serum,

respiratory secretions, cerebral spinal fluid,
urine, or some other body fluid.

If the antigen is present in the sample, it
will bind to the fixed antibodies.

In this example, the green shape represents
the antigen of interest and the other shapes

represent other molecules in the sample.

However, note that only the specific, or green,
antigen and none of the irrelevant molecules,

bind to the antibody-coated wells.

This accounts for the specificity of the test.

The wells are then washed out to remove any
of the unattached molecules leaving the antigen

of interest stuck to the wells.

Now a second antibody, directed against another
epitope on the target antigen is added.

These antibodies are conjugated covalently
to an enzyme indicated by the yellow circle

at the FC portion of the second antibody.

They bind to the antigen that's fixed in
the well and this provides a second level

of specificity for the assay.

The wells are washed again to remove any unbound
antibodies and in the final step, a solution

of a colorgenic enzyme substrate is added.

The interaction of the substrate and the captured
enzyme generates visible color in the

solution.

At the macroscopic level, the development
of color indicates those samples that have

second antibody bound to the target antigen
in the wells.

Thus, the wells that change color are the
ones that contain the antigen of interest,

in other words, the positive results.