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other diseases.
26
e
30
The autoantigen NY-ESO-1
was
biomarkers in diseases using this approach has
been limited to selected proteins. Because the rela-
tionship between protein structure, protein
expression, and antigenicity is not well under-
stood, there is a need for broad-based discovery
of autoantibodies without selection bias.
ed in esophageal cancer patient
sera and was later detected in the sera of patients
with many different cancers. These
first identi
findings led
to clinical trials targeting NY-ESO-1 for cancer
immunotherapy.
31
One key advantage of SEREX
is that the cDNA library expression re
ects the
expression levels, mutations, and splice varia-
tions that are inherent to the speci
PROTEOMICS METHODS FOR THE
DET
ECTION OF AUTOANTIBO
DIES
c tissues
from which the library is derived. However,
SEREX identi
cation of autoantigens has several
limitations. Full-length cDNAs are rarely cloned
into the expression vectors and truncated expres-
sion limits coverage. Many of the autoantibodies
that have been identi
Since the beginning of the 21st century, the
development and application of numerous pro-
teomics technologies has enabled high-
throughput protein expression for autoantigen
detection. Based on the source and type of
antigen repertoires, these technologies are
divided into four categories. Antigens have
been expressed as whole proteins from cDNA
libraries, separated and isolated from cell lysates
and displayed in protein array format, or
peptides can be displayed in microarray format
(see
Figure 1
). Each method displays unique
structural determinants and contributes to our
overall understanding of structural immunoge-
nicity (
Tables 1 and 2
).
ed target frame shift poly-
peptide products, which have yet not been
proven to be expressed at the protein level in the
relevant tissue. E. coli expression machinery has
poor expression of high molecular weight
proteins or proteins with multiple domains and
lacks chaperones to facilitate proper folding.
This limits the amount, type, and conformations
of proteins presented for screening. Plaque-
based screening is also not amenable to automa-
tion and quantitative analysis.
Several recent advances with phage-based
expression have resulted in the identi
cation of
multiple new autoantigens and have facilitated
automation. Lysates of individual E. coli colonies
have been spotted in microarray format for sero-
logic screening.
32
The use of
Phage Display of cDNA Libraries
After the development of immuno
uorescent
assays, serological
cation of recombi-
nantly expressed clones (SEREX), initially
described in 1995, represents one of the earliest
development of this approach that revolutionized
the discovery of autoantigens.
25
In this strategy,
cDNA libraries are generated from disease
tissues, cell lines, or testis tissue and the candidate
antigens are expressed either directly in E. coli or
using a lambda phage expression system. Colo-
nies or phage plaques containing expressed
proteins are then replicated onto nitrocellulose
membranes and probed with patient sera
(
Figure 1
, top). Positive clones are sequenced to
identify the autoantigens. This approachwas
identi
filamentous T7 or
M13 phage for protein expression results in
superior throughput compared with lambda
phage due to compatibility with biopanning.
33,34
More recently, solution-based phage display has
been developed to further identify autoantigens
expressed in a more native format. Phage display
is compatible with rapid biopanning for negative
and positive selection and has been integrated
with microarray platforms to improve high-
throughput multiplexing and quantitation. These
advances have led to the identi
cation of auto-
antibody biomarkers in several cancers such
as ovary and prostate.
33
e
35
In summary, the
primary advantages of phage-based expression
rst
applied to cancer but has been widely used for
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