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screened with sera for antigenicity. 54 e 56 Peptide
arrays can also be generated in vitro using on-
chip synthesis technology, similar to oligonucle-
otide Affymetrix arrays. 57 Peptides spanning the
proteome are
within sera, which is limited to antigens that
are present in sera and the availability of capture
antibodies for array printing. We, and others,
have developed in vitro transcription/translation
methods for expression and capture of target
tagged antigens onto an array format: nucleic
acid programmable protein arrays (NAPPA);
see Figure 2 . These arrays have the advantage of
real-time protein production for serologic detec-
tion. However, protein arrays in general have
limited PTMs and are limited by variable protein
expression, folding, and reproducibility. 45 e 48
A variation on spotting puri
ed bioinformatically.
DNA sequences encoding these peptides are
synthesized on-chip similar to DNA oligonucle-
otide arrays and expressed on the arrays using
in vitro T7-based expression systems. In compar-
ison to peptide-printed arrays, longer peptides
can be generated by in vitro synthesis. As an
alternative, the synthesized DNAs encoding the
peptide libraries can be displayed on the surface
of phage coat proteins for biopanning.
One key feature of antigen/peptide display for
antibody detection is that they lead to identi
first identi
ed proteins is to
spot mixtures of proteins that are produced in
multiwell plates using PCR ampli
ed genes and
in vitro expression system. 42,49,50
ca-
tion of the naturally occurring cognate antigen.
This identi
cation is critical for understanding
the pathogenesis of the disease. As a practical
alternative to developing biomarkers that are
speci
Peptide and Peptoid Arrays
One way to bypass the challenges of full-
length protein expression is to display peptides
rather than proteins. Peptides may be synthe-
sized in vitro as random peptides, peptide-like
structures (peptoids) or as peptides representing
the entire potential human ORFeome. Peptides
may be also be generated in vivo using phage,
E. coli, or yeast, cloned in fusion with a surface
protein, and displayed for biopanning. 51 e 53
With the development of massively parallel
peptide synthesis, tens of thousands of peptides
can now be synthesized,
c for diseases, display of peptide mimo-
topes or peptoids can be used to detect speci
c
antibodies in sera of patients with the disease.
Peptoids are similar to peptides but present
chemically synthesized diverse structural
epitopes for antibody selection. Peptoid microar-
rays displaying approximately 15,000 peptoids
have been successfully used for the identi
cation
s disease. 58
Although the initiating natural antigenic stim-
ulus cannot be identi
of autoantibodies
in Alzheimer
'
ed from the mimotopes,
immobilized, and
FIGURE 2 Serologic detection of
antibodies using programmable
protein arrays. Left: full-length
tagged cDNAs are printed on slides,
and detected with picogreen. Middle:
tagged antigens are expressed using
mammalian cell lysate, captured in situ
and protein expression is con
α
DNA
- GST
Case
Control
Add cell-free
expression
system
Add sera
rmed
using antitag antibodies. Right: arrays
are probed with sera, and bound
immunoglobulin is detected.
Detect IgG
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