Biology Reference
In-Depth Information
there is no need to label every primary antibody and one secondary
antibody can be used for a variety of primary antibodies. Even
more importantly, indirect immunolabeling offers signal amplifi ca-
tion ( see Subheading 1.4 ).
Primary antibodies are either polyclonal or monoclonal [ 6 ].
Polyclonal antibodies are collected from the serum of an immu-
nized animal (usually a herbivore) and recognize different epitopes,
thus increasing the likelihood of antibody binding. Unfortunately,
the serum will contain not only antibodies against the protein, pep-
tide, or carbohydrate used for immunization but also antibodies
against contaminant molecules and native antibodies of the immune
system, e.g., antibodies raised against the food which is especially
disturbing when working with plant cells. Therefore, pre-immune
serum should be collected prior to immunization and should be
used for control experiments. Monoclonal antibodies are produced
by cell cultures originating from fusion of an antibody-producing
cell with a myeloma cell and are specifi c for one epitope, thus
reducing unspecifi c binding. However, there is no guarantee that
this epitope is exposed and available for antibody binding.
Different classes of immunoglobulins are present in the serum
of immunized animals. Most antibodies used for immunolabeling
are IgGs which are purifi ed in order to reduce background stain-
ing. The antigen binding sites are localized at the light chains
located at the ends of the arms of the Y-shaped immunoglobulin.
1.3 Primary
Antibodies Used
for Immunolabeling
1.4 Secondary
Antibodies and Gold
Markers
The secondary antibody must specifi cally recognize the fi rst anti-
body; i.e., it must be directed against the host species of the pri-
mary antibody. For example, if the primary antibody is mouse IgG,
an anti-mouse immunoglobulin raised in another animal (e.g.,
goat) is required. The most frequently used secondary antibodies
are affi nity-purifi ed polyclonal IgGs (whole molecules) generated
by immunizing an animal with whole IgG. These secondary anti-
bodies recognize different epitopes so that several molecules with
their attached markers will bind to one primary antibody which
results in an amplifi cation of the signal as compared with direct
immunolabeling. Monoclonal secondary antibodies, fragments of
secondary antibodies, and antibodies raised against fragments are
likewise commercially available but not so often used for immuno-
labeling of plant cells.
Gold particles suitable for conjugation with antibodies are
available at sizes between 1.4 nm (nanoparticles) and 30 nm. The
larger gold particles can be seen at low magnifi cations of the EM.
They have the disadvantage, however, that because of steric
hindrance, a lower number of secondary antibodies can bind to the
primary antibody which decreases the extent of signal amplifi ca-
tion. Therefore, it is better to use antibodies conjugated to small
particles and, if necessary, to increase their size by silver
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