Measuring Serum Antibody with an Agglutination Assay

This short animation demonstrates measuring serum antibody with an 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 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.