Agglutination assay to detect antigens

This short animation demonstrates detection of specific antigens using the agglutination assay. 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.) Agglutination assays have been used for decades as a simple method to detect antigenic substances

in biologic samples.

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

its limitations.

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

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

detect.

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

surface.

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

on the slide.

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

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

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

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

of interest.

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

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

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

milky in appearance to clear and transparent.

This transition should occur in the area with
the positive control.

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

The negative control circle should remain
unclumped and opaque.

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

bind to numerous antigens.

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

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

that have repetitive antigenic domains.

Molecules like microbial capsules, flagella,
or lipopolysaccharides.

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

together or, agglutinate.

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

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

This is the basis of the test.

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