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The
enzym-linked immuno spot
assay (ELISPOT) is probably
the oldest of the alternatives we would like to introduce. It is a variation of
the enzyme-linked immuno
sorbent assay, the ELISA. Antibodies that recognize the target
antigen are
coated in a 'culture' plate. This can be a cyto- or chemokine, but
it might also be an antibody or receptor antagonist. Cells are stimulated in the coated wells and after the incubation period washed
away. A second, enzyme-labeled antibody is added to the wells and the excess
(non-binding) antibody is washed away again. The last incubation step is the
addition of a chromogen, which will precipitate on the place (spot) of producing
cell. When the density of producing cells is not too high the frequency of
producing cells to the total number of cells can be calculated. When the
frequency of the positive cell is too high the spots will merge and accurate
counting will not be possible.
This method is a very simple and relatively cheap technique that doesn't require
expensive apparatus, except for a reversed (culture) microscope. Computerized
readers make the time consuming evaluation less laborious. Detecting more than two different molecules in one well
is difficult. Detection of the molecule together with the phenotype of the
producing cells is also 'not' possible. The ELISPOT can be the solution to detect antigens that are difficult to detect
by intracellular staining though.
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ELISPOT
