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LABORATORY TECHNIQUES IN
IMMUNOLOGYLaboratory techniques in immunologyVarious laboratory techniques exist that rely on the use of
antibodies to visualize components of microorganismsor
other cell types and to distinguish one cell or organism type
from another.
Electrophoresisis a technique whereby the protein or
carbohydrate components of microorganisms can be separated
based upon their migration through a gel support under the
driving influence of electricity. Depending upon the composi-
tion of the gel, separation can be based on the net charge of the
components or on their size. Once the components are sepa-
rated, they can be distinguished immunologically. This appli-
cation is termed immunoelectrophoresis.
Immunoelectrophoresis relies upon the exposure of the
separated components in the gel to a solution that contains an
antibodythat has been produced to one of the separated pro-
teins. Typically, the antibody is generated by the injection of
the purified protein into an animal such as a rabbit. For exam-
ple, the protein that comprises the flagellar appendage of a
certain bacteriacan be purified and injected into the rabbit, so
as to produce rabbit anti-flagellar protein.
Immunoelectrophoresis can be used in a clinical
immunologylaboratory in order to diagnose illness, especially
those that alter the immunoglobulin composition of body
fluids. Research immunology laboratories also employ immu-
noelectrophoresis to analyze the components of organisms,
including microorganisms.
One example of an immunoelectrophoretic technique
used with microorganisms is known as the Western Blot.
Proteins that have been separated on a certain type of gel sup-
port can be electrically transferred to a special membrane.
Application of the antibody will produce binding between the
antibody and the corresponding antigen. Then, an antibody
generated to the primary antibody (for example, goat anti-rab-
bit antibody) is added. The secondary antibody will bind to theprimary antibody. Finally, the secondary antibody can be con-
structed so that a probe binds to the antibody’s free end. A
chemical reaction produces a color change in the probe. Thus,
bound primary antibody is visualized by the development of a
dark band on the support membrane containing the elec-
trophoretically separated proteins. Various controls can be
invoked to ensure that this reaction is real and not the result of
an experimental anomaly.
A similar reaction can be used to detect antigen in sec-
tions of biological material. This application is known as
immunohistochemistry. The sections can be examined using
either an electron microscopeor a light microscope. The
preparation techniques differ for the two applications, but
both are similar in that they ensure that the antigen is free to
bind the added antibody. Preservation of the antigen binding
capacity is a delicate operation, and one that requires a skilled
technician. The binding is visualized as a color reaction under
light microscopic illumination or as an increased electron
dense area under the electron beam of the electron micro-
scope.
The binding between antigen and antibody can be
enhanced in light microscopic immunohistochemistry by the
exposure of the specimen to heat. Typically a microwave is
used. The heat energy changes the configuration of the antigen
slightly, to ease the fit of the antigen with the antibody.
However, the shape change must not be too great or the anti-
body will not recognize the altered antigen molecule.
Another well-establish laboratory immunological
technique is known as enzyme-linked immunosorbent assay.
The technique is typically shortened to ELISA. In the ELISA
technique, antigen is added to a solid support. Antibody is
flooded over the support. Where an antibody recognizes a
corresponding antigen, binding of the two will occur. Next
an antibody raised against the primary antibody is applied,
and binding of the secondary antibody to the primary mole-
cule occurs. Finally, a substrate is bound to a free portion of
the secondary antibody, and the binding can be subsequently
visualized as a color reaction. Typically, the ELISA test iswomi_L 5/6/03 3:31 PM Page 333