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(English captions by Andrea Matsumoto, University of Michigan.) Many simple rapid diagnostic tests detect[br]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[br]how a prototype of this assay works.

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The enzyme immunoassay can be done in a multi-well[br]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[br]so, lets take a closer look at one of the wells

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of this assay to see what happens during the[br]performance of the assay.

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The plate is prepared to perform a particular[br]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[br]sample, which could be a sample of serum,

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

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

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In this example, the green shape represents[br]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,[br]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[br]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[br]epitope on the target antigen is added.

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These antibodies are conjugated covalently[br]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[br]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[br]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[br]enzyme generates visible color in the

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solution.

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

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second antibody bound to the target antigen[br]in the wells.

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

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in other words, the positive results.