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(English captions by Andrea Matsumoto, University of Michigan.) Agglutination assays have been used for decades as a simple method to detect antigenic substances

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in biologic samples.

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The purpose of this video is to explain how[br]this method works in practice and to expose

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its limitations.

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The agglutination assay uses tiny particles,[br]most often latex beads.

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The beads are coated with a specific antibody[br]against the antigen that you would like to

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detect.

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The test is usually performed on a card or,[br]glass or plastic slide, often one with a black

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surface.

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First you add a suspension of the coated latex[br]beads to each of the three encircled areas

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on the slide.

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Note that the suspension is concentrated enough[br]to produce a milky appearance on the background.

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Now you add a few drops of the unknown sample[br]that you are interested testing.

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But, you will also need to use one circled[br]area for a negative control solution that

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contains no antigen and another for a positive[br]control solution that contains the antigen

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of interest.

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Next the slide is gently rocked or swirled[br]to mix the beads with the test solutions and

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the samples containing the antigen of interest[br]will begin to agglutinate the beads.

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This will produce the appearance of visible[br]clumps and the solution itself will turn from

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milky in appearance to clear and transparent.

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This transition should occur in the area with[br]the positive control.

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If the antigen is present in the unknown sample[br]then it will form clumps.

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The negative control circle should remain[br]unclumped and opaque.

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Recall that the latex beads are coated with[br]a specific antibody so that each bead can

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bind to numerous antigens.

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For agglutination to work the antigen of interest[br]must also be able to bind to multiple beads.

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Therefore in this assay, antigens that can[br]be detected are limited to large macromolecules

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that have repetitive antigenic domains.

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Molecules like microbial capsules, flagella,[br]or lipopolysaccharides.

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One long repeating antigen molecule can then[br]attach to several beads causing them to clump

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together or, agglutinate.

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So even very tiny quantities of antigens that[br]have lots of repeating antigenic domains can

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cause visible clumps to form and be detected[br]by this test.

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This is the basis of the test.

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Finally here are some examples of agglutination[br]assays that are used in clinical practice.