(English captions by Andrea Matumoto, University of Michigan.) An agglutination assay is a simple way to detect and to measure antibodies in a clinical
specimen directed against a specific antigen of interest.
In this animation the principles and potential pitfalls of the assay will be demonstrated.
The main reagent used in the assay is a solution of insoluble tiny beads usually composed of
latex.
Alternatively to measure antibodies against a microbial pathogen the killed bacterial or
yeast cells can be used as the agglutinating particle.
However in this example latex beads are used and they have been prepared so that the antigen
of interest coats their surfaces and they are concentrated enough to produce a visible
milky suspension.
To measure antibodies against the antigen the particles are added to the wells of a
ninety-six well microtiter plate and then sera taken from different patients are added
to the wells in the first column.
Two fold dilutions of the sera are prepared in the rows and then a known negative and
positive control serum are added in the last two columns.
When the plate is allowed to incubate at room temperature the wells containing the negative
control serum remain unchanged.
The wells containing the positive control serum have developed a visible button at the
bottom of the wells and the solution in those wells has changed from milky to clear.
So what accounts for the appearance of the positive wells and what accounts for the lack
of change in the negative wells?
To understand what's going on, lets take a microscopic look at the negative and positive
wells to see what is happening in each case.
When there is no antibody in the well with the beads they remain in suspension giving
the well a milky appearance.
However, when specific antibody is present it binds to and crosslinks the beads.
This causes the beads to clump and to form large aggregates that sink to the bottom of
the round bottom wells.
They make the suspension clear as they sink instead of milky and the aggregates settle
into a pellet or a button which forms at the bottom of the well.
So you will see the button at the bottom of any well that has enough specific antibodies
in it to precipitate the beads.
But when the antibody is diluted out, the button no longer appears.
The patient's antibody titer is the last dilution of serum that produces a button.
But how to we explain the absence of a button in the most concentrated wells of the serum
with the highest titer showed by the orange arrow?
Imagine a well that has many more antibody molecules in it than beads.
In this situation the beads will be completely coated with antibody and there will be no
possibility of crosslinking and precipitation.
This phenomenon, which is referred to as a prozone sometimes occurs in cases of syphilis.
The standard screening test for syphilis is an agglutination assay in which undiluted
serum is added to beads coated with the antigen cardiolipin.
In secondary syphilis the antibody titers are sometimes so high that the test exhibits
a prozone and is falsely negative.
So how do you think you could overcome this potential problem and make the correct laboratory
diagnosis?
Did you think of diluting the serum and then retesting it?
By diluting the antibody in this situation the amounts of antibody and antigen are closer
to being equivalent with one another.
Then the conditions for crosslinking can exist.
Now when crosslinking occurs a precipitate forms and the button develops at the bottom
of a tube indicating a positive test.
Subtitles by the Amara.org community