ANTIBODY-BASED TECHNIQUES - A Guide to Methods in the Biomedical Sciences - page 80

Enzyme-linked immunospot (ELISPOT) assays

The ELISPOT assay was originally developed as an alternative to
the plaque assay (34) developed by Niels Jerne (who won the Nobel
Prize for his theories on the specificity of the immune system in 1984).
The plaque assay was developed as a method to detect and quantitate
individual antibody secreting cells. This assay limited the range of anti-
body specificities that could be measured, and relied on an additional
step of adding fresh serum (as a source of complement) to produce
the characteristic plaques. The ELISPOT was developed to circumvent
these issues. With improvements in the assay, it is now one of the two
methods used to enumerate cytokine producing cells. A cytokine is a
small protein molecule produced by cells of the immune system that
regulates other immune cells. Many cytokines provide communication
between the immune system and other cells of the body as well. With
the use of high affinity antibodies, ELISPOT assays can detect cells
that produce as few as 100 molecules of a protein per cell per second.
Compare that with an antibody producing cell, which produces 1000 to
10,000 antibody molecules per cell per second.
The ELISPOT assay starts with a membrane (usually nitrocellulose)
coated with a specific antibody against, for example, the cytokine of in-
terest. The membrane is placed in microtiter wells. Cells are then added
to the well and incubated overnight. A second antibody to the cytokine
is added (coupled with an enzyme) and its presence is detected by ad-
dition of a substrate. The development of “spots” identifies the location
of a single, cytokine producing cell.

FACS

FACS (see below) can also be used to identify cytokine producing
cells. Cells are incubated to allow cytokine production as for ELISPOT.
However, because FACS is a cell-based assay, an agent is added during
incubation which allows cytokine production but prevents its secretion.
Brefeldin A is commonly used for this purpose, which prevents forward
trafficking and secretion of newly synthesized proteins. The cells can be
permeabilized and stained with a fluorescenated anti-cytokine antibody.

D. Flow cytometry and fluorescence activated

cell sorting (FACS)

FACS is a method that allows the separation of different cell pop-
ulations or organelles to be analyzed and isolated using fluorescently
tagged antibodies. FACS was developed in the 1960's by Leonard A.