466
Autoantibodies and Autoimmunity
have multiple autoantibody specifcities to
nucleolar autoantigens that are unrelated
at the macromolecular level (i.e. not part
oF the same macromolecular complex).
3
Autoantibodies as Molecular and Cellular
Probes
Autoantibodies can be used For the de-
tection
oF
their
cognate
antigens
us-
ing immunoprecipitation, immunoblot-
ting, enzyme-linked immunosorbent as-
say (ELISA), and a variety oF microscopy
techniques including immunoelectron mi-
croscopy. The most visually impressive
demonstration oF the useFulness oF au-
toantibodies as biological probes is the
indirect immunofluorescence (II±) test.
In this technique (±ig. 2), a cell or tis-
sue source containing the autoantigen oF
interest is permeabilized, to allow entry
oF the antibody into the cell, and fxed,
to ensure that the target antigen is not
leached away during the procedure. Al-
though some procedures are inappropriate
For particular antigens, workable means
oF cell permeabilization and fxation have
been developed. The cell substrate is in-
cubated with the autoantibody to allow
interaction with the antigen, and any ex-
cess is washed away. The location oF
the antigen/autoantibody complex within
the cell is revealed by addition oF an
anti-antibody tagged with a fluorochrome.
±luorescence microscopy is then used to
view the cells to determine the location oF
the antigen-/autoantibody-/fluorochrome-
tagged anti-antibody complex. Using this
technique, investigators are identiFying
an increasing number oF autoantibody
specifcities that recognize cellular sub-
structures and domains (Table 2, ±igs. 3
and 4). The nucleus can be identifed by
a variety oF autoantibodies such as those
against chromatin and DNA or, as shown
in ±ig. 3(a), autoantibodies to the nuclear
lamina, which underlies the nuclear enve-
lope and produces a ringlike fluorescence
around the nucleus. The nucleolus and its
subdomains can also be identifed by a va-
riety oF autoantibody specifcities (Table 2).
Autoantibodies against the 34 kDa protein
fbrillarin, a component oF the C/D box
containing small nucleolar ribonucleopro-
tein (snoRNP) particles, label the nucleolus
in a distinctive ‘‘clumpy’’ pattern (±ig. 3e).
The list oF autoantibodies that are able
to distinguish subnuclear domains and
compartments, some considerably smaller
than the nucleolus, continues to grow.
One example is the Cajal body, a small
subnuclear structure described using light
microscopy by the Spanish cytologist San-
tiago Ramon y Cajal in 1903 and sub-
sequently named aFter him. This nuclear
domain can now be easily identifed us-
ing human autoimmune sera that react
with p80 coilin (±ig. 3d), a protein highly
enriched in the Cajal body. Using other au-
toantibodies, it has been Found that Cajal
bodies contain snRNP particles and fbril-
larin (previously thought to be restricted
to the nucleolus and prenucleolar bodies).
Knowledge oF the Functional associations
oF these coiled-body constituents suggests
that the Cajal body may play a role in RNA
processing and/or in the accumulation oF
components involved in RNA processing.
Many Features oF subcellular structures
such as size, shape, and distribution can
be studied by II± during the cell cycle, vi-
ral inFection, mitogenesis, or any cellular
response that may result in changes in the
distribution oF an antigen or a subcellular
structure. As shown in ±ig. 3(a) (arrow-
heads), antinuclear lamin autoantibodies
can be used to reveal re-Formation oF the
lamina during telophase. Autoantibodies
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