How Does This Work?
The interaction of antigen and antibody outside the body--in the laboratory--can be
used to determine if a patient has an infectious or an autoimmune disease. The
test measures whether a specific antibody associated with an illness can be found
in a patient's blood. A positive result indicates that the antibody is there and
implies that the person has encountered a particular disease.
This exercise begins with removing red and white blood cells, which can interfere
with the test, from a patient's blood sample. The watery fluid that remains is
called serum. A portion of serum possibly containing the antibody is allowed to
react with the target antigen. A correct match causes the antigen and antibody to
Detection becomes possible when a second antibody is added. This antibody is
prepared from the serum of an animal injected previously with human antibody; the
human antibody in this case serves as an antigen and the animal thus produces an
antibody against the human antibody. Once isolated, the second antibody can be
chemically linked to a system that can produce a detectable signal.
In ELISAs, the antigen antibody complex is exposed to the second antibody,
which binds to the antibody portion of the complex (against which it was formed),
creating a sandwich-type structure (Figure 1). The signaling system consists of
an enzyme attached to the second antibody. When the appropriate chemical is
added, the enzyme converts it to a colored substance that can be measured.
This test quantifies how much enzyme is present by the amount of color produced.
The more enzyme present, the more secondary antibody must be attached. The amount
of secondary antibody present is determined by the amount of target, or first
antibody, available. Finally, because the first antibody binds to antigen, the
more antigen that is accessible, the more first antibody will be retained. The
measure of color, therefore, reflects the amount of antigen initially present.
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