1
00:00:00,000 --> 00:00:06,000
(English captions by Andrea Matsumoto, University of Michigan.) Agglutination assays have been used for decades as a simple method to detect antigenic substances

2
00:00:06,000 --> 00:00:07,000
in biologic samples.

3
00:00:07,000 --> 00:00:12,000
The purpose of this video is to explain how
this method works in practice and to expose

4
00:00:12,000 --> 00:00:15,000
its limitations.

5
00:00:15,000 --> 00:00:20,000
The agglutination assay uses tiny particles,
most often latex beads.

6
00:00:20,000 --> 00:00:24,000
The beads are coated with a specific antibody
against the antigen that you would like to

7
00:00:24,000 --> 00:00:27,000
detect.

8
00:00:27,000 --> 00:00:33,000
The test is usually performed on a card or,
glass or plastic slide, often one with a black

9
00:00:33,000 --> 00:00:34,000
surface.

10
00:00:34,000 --> 00:00:39,000
First you add a suspension of the coated latex
beads to each of the three encircled areas

11
00:00:39,000 --> 00:00:41,000
on the slide.

12
00:00:41,000 --> 00:00:49,000
Note that the suspension is concentrated enough
to produce a milky appearance on the background.

13
00:00:49,000 --> 00:00:53,000
Now you add a few drops of the unknown sample
that you are interested testing.

14
00:00:53,000 --> 00:00:59,000
But, you will also need to use one circled
area for a negative control solution that

15
00:00:59,000 --> 00:01:04,000
contains no antigen and another for a positive
control solution that contains the antigen

16
00:01:04,000 --> 00:01:12,000
of interest.

17
00:01:12,000 --> 00:01:20,000
Next the slide is gently rocked or swirled
to mix the beads with the test solutions and

18
00:01:20,000 --> 00:01:25,000
the samples containing the antigen of interest
will begin to agglutinate the beads.

19
00:01:25,000 --> 00:01:29,000
This will produce the appearance of visible
clumps and the solution itself will turn from

20
00:01:29,000 --> 00:01:34,000
milky in appearance to clear and transparent.

21
00:01:34,000 --> 00:01:36,000
This transition should occur in the area with
the positive control.

22
00:01:36,000 --> 00:01:41,000
If the antigen is present in the unknown sample
then it will form clumps.

23
00:01:41,000 --> 00:01:47,000
The negative control circle should remain
unclumped and opaque.

24
00:01:47,000 --> 00:01:52,000
Recall that the latex beads are coated with
a specific antibody so that each bead can

25
00:01:52,000 --> 00:01:54,000
bind to numerous antigens.

26
00:01:54,000 --> 00:02:00,000
For agglutination to work the antigen of interest
must also be able to bind to multiple beads.

27
00:02:00,000 --> 00:02:05,000
Therefore in this assay, antigens that can
be detected are limited to large macromolecules

28
00:02:05,000 --> 00:02:09,000
that have repetitive antigenic domains.

29
00:02:09,000 --> 00:02:15,000
Molecules like microbial capsules, flagella,
or lipopolysaccharides.

30
00:02:15,000 --> 00:02:20,000
One long repeating antigen molecule can then
attach to several beads causing them to clump

31
00:02:20,000 --> 00:02:23,000
together or, agglutinate.

32
00:02:23,000 --> 00:02:27,000
So even very tiny quantities of antigens that
have lots of repeating antigenic domains can

33
00:02:27,000 --> 00:02:32,000
cause visible clumps to form and be detected
by this test.

34
00:02:32,000 --> 00:02:35,000
This is the basis of the test.

35
00:02:35,000 --> 00:02:39,000
Finally here are some examples of agglutination
assays that are used in clinical practice.