Enzyme-Linked Immunosorbent Assay (ELISA)

This short animation demonstrates enzyme-linked immunosorbent assay (ELISA) to measure specific antibodies. 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.) The enzyme-linked immunosorbent assay or ELISA is a commonly used format for serologic testing.

The purpose of this animation is to explain
how this test works.

ELISA serologies are usually done in multi-well
microtiter plates so that dilutions of serum

can be easily prepared and tested.

To better understand how this assay is done,
lets take a closer look at what happens in

one of the wells of this assay plate.

To perform the assay, the wells are coated
with the antigen of interest.

For commercial tests, this would be done by
the manufacturer of the assay.

To begin testing, the wells are filled with
dilutions of the patient's serum.

If antibodies against the antigen are present
they will bind to the antigen fixed to the

bottom of the wells.

But only antigen specific antibodies will
bind to the wells.

The wells are then washed out to remove all
unbound antibodies.

Next a solution of an animal antibody against
human antibodies is added.

This second antibody is covalently conjugated
to an enzyme.

The wells are washed again, this time to remove
any unbound enzyme conjugated antibody.

Finally a solution of a colorgenic enzyme
substrate is added.

The interaction of the substrate with the
enzyme on the second antibody generates visible

color.

The development of color in the wells of the
specific antibody can be seen with the naked

eye or quantified with an electronic plate
reader.

The reaction is less intense as the serum
is diluted and the amount of antibody captured

in the wells decreases.

The titer is the highest dilution with definite
color development.