In the direct immunofluorescence test, there is a biopsy
section or other tissue section placed on a slide.
A fluorescein-labeled sheep antibody
against the antigen of interest is added.
So for example, one might want to see whether a particular
patient in a biopsy was expressing a particular tumor antigen.
And one could use a sheep antibody
specific for that tumor antigen.
Under ultraviolet light, the fluorescein label
emits a visible light over the antigen of interest.
So for example, if we are looking for
a specific tumor antigen, then if
the patient expresses that tumor
antigen, there will be light emission.
If they do not, there
will be no light emission.
In the indirect immunofluorescence test,
the tissue can be of animal or human
origin as long as it contains the
antigen that one is interested in.
Patient serum is added, and in this
particular example where we are
looking for autoantibodies, the
autoantibodies bind to the antigen.
So this could be a tissue section of
thyroid for example, and we’re asking
the question: does the patient have
autoantibodies to thyroid antigens?
A fluorescein-labeled sheep anti-human IgG is added
and this binds to the patient’s immunoglobulin gene.
The bound IgG is detected
under ultraviolet light.
One example of this would also be
the measurement of anti-nuclear
antibodies in patients with systemic lupus erythematosus.
Here we have patient’s serum that
is incubated with a section of
rat liver, a section of rat kidney and a section of rat stomach.
Binding to the antigens present in these tissues is detected
by a fluorescein labeled anti-human immunoglobulin antibody.
And we can see the patient is indeed positive
for these anti-nuclear antibodies where we
can see fluorescence of a liver section, of
a kidney section and of a stomach section.
As well as these immunofluorescence techniques,
one can also use immunohistochemistry,
where a tissue section on a glass slide is
incubated with an enzyme-labeled antibody.
Binding will occur if the antibody
detects antigens in the tissue section.
The substrate for the
enzyme can be added.
The enzyme mediates a color
change in this substrate.
And this can be detected
under the microscope.
In indirect immunochemistry, the patient’s antibody is
detected using an enzyme labeled anti-human immunoglobulin.
Again the enzyme mediates a color change in the
substrate which can be visualized under a microscope.