WEBVTT

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

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using an enzyme immunoassay.

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

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

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surface.

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

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

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

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that bind to the antigen of interest.

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

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

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

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

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represent other molecules in the sample.

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

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bind to the antibody-coated wells.

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This accounts for the specificity of the test.

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

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of interest stuck to the wells.

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

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

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at the FC portion of the second antibody.

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

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of specificity for the assay.

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

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of a colorgenic enzyme substrate is added.

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

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solution.

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

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second antibody bound to the target antigen
in the wells.

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

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in other words, the positive results.