ELISA - Enzyme Linked Immunosorbent Assay, is a very,
very commonly employed analytical method in immunology.
Plastic plates are
coated with an antigen.
They can be coated with an antibody as well, but in this
particular example we are coating the plates with an antigen.
Very often one will purchase already
coated plates from a manufacturer.
The excess antigen is washed off.
Test antibody is then added.
So maybe this is an autoantigen
that is coated on the plate.
And we want to discover whether a patient
has autoantibodies against that autoantigen.
If they do, then they will bind, those
autoantibodies will bind to the autoantigen.
Following a period of
incubation, the plate is washed.
And then a ligand to the
test antibody is added.
In this case the ligand is another antibody
coupled to the enzyme horseradish peroxidase.
The plate is again washed and a substrate, a chromagen is added
and a colored product is produced by the enzyme reaction.
The amount of color is proportional to the
amount of antibody bound to the antigen.
In a sandwich immunoassay, first
of all antibody is bound to the
well of a microtitre plate, which is typically a 96 well plate.
Varying amounts of antigen are added.
Any unbound antigen is
then removed by washing.
So for example, maybe we want to measure the amount
of a particular substance in the serum of a patient.
This will be detected
by a specific antibody.
Following washing, a labeled second antibody is added that
is specific for a non-overlapping epitope of the antigen.
So let’s say we are trying
to measure a cytokine.
The first antibody coating the plate would be
for one epitope on that particular cytokine.
The other antibody would be for a different
epitope, a different part of the same cytokine.
Labels include radioactive isotopes or an enzyme that
causes a substrate to change color or emit light.
This would be in a
The unbound labeled second
antibody is removed by washing.
And then the amount of second
antibody bound is measured.
What is able to determine the amount
of bound second antibody as a function
of the concentration of antigen added
with construction of a standard curve.
In the fluorescent microsphere Luminex assay, there
are beads that are coated with particular antigens.
Patient’s serum is added to the
coated beads and then incubated.
There is a reporter tag, for example
fluorescently labeled anti-human IgG antibody
added to detect the binding of the patient’s
antibody to the antigen coating the bead.
These beads are then subject to a
technique called flow cytometry,
and signals that identify each bead are emitted and detected.
There is also a second signal
that identifies the reporter tag.
The amount of signal is proportional to in this example,
anti-hepatitis A antibody in the patient’s serum.
So in this particular example, we have
beads coated with different antigens from
Hepatitis A, B and C and one can detect that
the patient has antibodies to Hepatitis A.