Biomedical Engineering Reference
In-Depth Information
as in the same way that cold competitors compete for binding in EMSA;
see Chapter 3). This allows for the establishment of a standard curve,
in which the ratio of the bound antigen/free antigen is plotted versus
the amount of unlabeled antigen added, where the bound/free is de-
termined by counting the radioactivity associated with the antibody and
that released from the antibody (there are a number of ways of separat-
ing these depending on how the assay is initially set up). By comparing
the amount of radioactive antigen displaced by an “unknown” sample,
the concentration of the antigen in the sample can be determined with
accuracy.
Most radioimmunoassay employ the use of antigens labeled with ra-
dioactive iodine
but tritium
antigens are also some-
times used.
Enzyme-linked immunosorbent assay (ELISA)
The ELISA was first described by Engvall and Perlman in 1971 (33).
ELISA is the most commonly used method for analysis of soluble anti-
gens and antibodies. ELISA methods lend themselves to automation,
making the quantitation of proteins in a large number of samples possi-
ble with relative ease. ELISAs are also highly sensitive, on par with RIA,
do not involve the use of radioactivity, and can be used in a variety of
permutations. In the most basic method, to quantitate an antibody re-
sponse, wells of microtiter plates (96 well plates) are first coated with an
antigen. Then serum samples (if an animal or person has been immu-
nized) or supernatants (for example from tissue culture wells in which
monoclonal antibodies are being screened) are added to the coated
wells. After washing, a developing secondary antibody is added. Sec-
ondary antibodies are those that have been made in one species that
recognize antibodies form a different species (for example, goat anti-
mouse antibodies, or rabbit ant-goat antibodies, etc.) The secondary
antibody is coupled to an enzyme (hence, the name “enzyme-inked”).
The conversion of substrate is directly proportional to the amount of
bound secondary antibody, which in turn is proportional to the amount
of antibody bound to the antigen.
A second type of ELISA is the sandwich ELISA. In a sandwich ELISA,
the plate is first coated with an antibody to the antigen, and the antigen
added. This assay is particularly useful when pure forms of the antigen
are not available. The remainder of the procedure is the same as out-
lined above. In general, sandwich ELISAs are the most sensitive and
can detect proteins in concentrations of 100 pg/ml to 1 ng/ml to
grams). Standard ELISAs are generally one order of magnitude
less sensitive.
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