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them from antibodies produced by our immune
system when encountering foreign antigens.
Genomic sequencing has identi
Progressive Purification of Antigens
Identi
cation of autoantigens has paralleled
the technological development in immuno-
assays over the past six decades, from crude
cellular
ed approxi-
mately 20,000 unique open reading frames in
the human genome as source antigenic struc-
tures. Tissue-speci
fractions
14,15
to
chromatographic
c splice variation and poly-
morphisms add signi
peaks,
16
gel bands,
17
e
20
and the identi
cation
cant variation to the
complexity of self-antigens. As a result, our
understanding of immune responses requires
a systems immunology proteomics approach
that can produce thousands of protein structures
to identify antibody signatures that are associ-
ated with disease. In this chapter, we focus on
the application of diverse proteomic technolo-
gies for the discovery of autoantigens.
of protein structures. One of the
first autoanti-
bodies identi
ed was rheumatoid factor (RF),
an antibody against the Fc domain of human
immunoglobulins. Targets of ANAs were identi-
fied using crude characterizations of antigens in
cellular/nuclear extracts by immunodiffusion,
probing with sera from patients with Sjogren
s
syndrome and SLE. This work led to the
discovery of several well-known antigens such
as Ro/SSA, La/SSB, Sm, RNP, Scl-70, and Jo-1,
which are intracellular antigens complexed
with nucleic acids.
21
Development of protein
electrophoresis, immunoblotting, and immuno-
precipitation techniques provided tools to re
'
History of the Detection
of Autoantibodies
ne
our knowledge of autoantigens to the level of
molecular weight. It was not until the advent
of molecular biology that the exact autoantigens
could be identi
Immunofluorescent Assays
The development of techniques for the detec-
tion of autoantibodies has focused on diseases
associated with autoimmunity, where a direct
relation of autoantibody titer to disease patho-
genesis has been established. The presence of
disease-speci
ed at the molecular level.
Targeted Detection of Autoantibodies
Initially, candidate autoantigens were chosen
based on prior experimental and/or theoretical
knowledge of their potential roles indiseasedevel-
opment. Inparticular, oncogenes that are critical to
cancer development and overexpressed in cancers
were selected for discovery of autoantibodies.
These oncogenes were puri
c antibodies in the sera of patients
with autoimmune diseases was
first described
more than half a century ago.
1
e
6
Initial studies
of sera from patients with systemic lupus ery-
thematosus (SLE) led to the discovery of the
major class of autoantibodies, antinuclear anti-
bodies (ANAs).
7,8
ANAs were initially detected
with immuno
uorescent assays (IFA) using
cultured cells probed with patient sera,
9
e
13
but
identi
ed as recombinant
proteins from E. coli and tested in ELISA or
Western blot for antibodies in the sera of cancer
patients. This hypothesis-driven approach led
to the identi
c targets of these
ANAs has been limited. Molecular characteriza-
tion of autoantigens is required to transform
these cell-based semiquantitative assays into
reproducible, quantitative assays to improve
diagnosis and disease monitoring. Determining
the identities of these autoantigens will further
our understanding of the underlying mecha-
nisms of disease pathogenesis in autoimmunity
and devise therapeutics for disease management.
cation of
the speci
cation of autoantibodies against
several oncogenes in cancers. For example, anti-
c-myc autoantibodies were identi
ed by Western
blot in 25 out of 44 sera from patients with colo-
rectal cancer, but in 8 out 46 normal donors.
22
Autoantibodies against other oncogenes such as
ras or HER-2/neu have also been reported.
23,24
However,
the
discovery
of
autoantibody
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