Download Epitope mapping of humoral immune response

Detailed epitope mapping of humoral immune response towards persisting
Epstein-Barr virus infections using peptide microarrays
Paul von Hoegen, Nikolaus Pawlowski, Janina Seznec, Ulf Reimer
JPT Peptide Technologies, Berlin, Germany.
Introduction
Schematic
layout
All of us are infected by a number of viruses causing
persistent infections and staying in our bodies for a long
period of time, sometimes the whole life. While, in most
healthy individuals not recognized, persistent viral
infections can cause a number of serious health
problems. EBV, for instance, the causative agent of
infectious mononucleosis infects over 90% of the human
population. The virus is thought to contribute to diseases
such as different cancer types, multiple sclerosis and
chronic fatigue syndrome. Probably, all of us feel
sometimes infected. Additionally, persistent infections are
a critical issue in organ and stem cell transplantation.
The correlation of persistent infections with pathologies is
an important issue; it is the gate to new diagnostics and
therapies. However, passing the gate is still a demanding
task. The detailed epitope mapping of humoral immune
response in human serum samples allows a high
resolution analysis of the antibody repertoire against EBV
antigens. This information can then be correlated with
clinical phenotypes. We developed a flexible peptide
microarray platform which allows the presentation of up to
6900 peptides on a single microscope glass slide.
Peptides are immobilized chemoselectively and directed
onto the surface. A further highlight of the platform is the
low volume of sample required; 1µl is enough. Serum
incubations and data evaluation can be carried out using
standard DNA-microarray equipment.
We show examples where this approach allows to
differentiate different donors for anti EBV humoral
immune responses.
QC-scan
Image after
incubation
with serum
SA1
Results
• Individual signal patterns are observed for immunogenic
proteins.
• Signals span along the peptide scan
VP26
SA2
SISSLRAATSGATAA
VSSSISSLRAATSGA
TPSVSSSISSLRAAT
SA3
ALASSAPSTAVAQSA
GTGALASSAPSTAVA
ASAGTGALASSAPST
QAAASAGTGALASSA
Fig. 2. Layout of peptide microarray (left) and images after
printing (QC-scan, center) and after serum incubation (right).
Three subarrays are used for improved data quality.
Fig. 5. Signal patterns for peptide scan
through EBV capsid protein VP26 for
three individuals. The shared residues
in the overlapping peptide indicate the
linear epitope recognized by antibodies
of donor 3.
Seroscreening
• Screening of sera of healthy volunteers
• Evaluation of images using GenePix
• Processing of data and calculation for QC with R
Data Quality
SA1
SA2
SA3
Library Design
• Overlapping peptide scans through major EBV antigens
(BLRF2, BZLF1, EBNA1, EBNA3, EBNA4, EBNA6,
LMP1, VP26) (1465 peptides)
• Follow-up library to include sequence variants and
posttranslational modifications.
Array Production
Synthesis of peptide with
N-terminal reactivity tag
Library generation
SPOTsynthesis
QC-Scan &
post-printing
procedures
Cleavage &
reformatting
Chemoselective
immobilization
on microarray slides
Fig. 6. Patterns of antibody reactivities in human serum samples
towards EBV antigens. Donor 1 shows antibody reactivity against
EBNA1, donor 2 against EBNA1 and EBNA6 and donor 3 shows
reactivity against almost all antigens except BLRF2 and EBNA6.
Fig. 3. Typical intra array reproducibility between the three
subarrays.
Fig. 4. Reproducibility between different assays for one
sample. The scatterplots show
the mean signals per peptide
in three independent experiments.
• Development of a high content and high throughput
screening platform for biomarker discovery
• Screening of low volume serum samples results in specific
signal patterns1,2,3
• Signal patterns for persistent infection can be compared to
clinical data
• Library can be easily extended to sequence variants and
posttranslational modifications.
References
1
Printing
process
2
3
Fig. 1. Schematic representation of the array production process.
* Correspondence should be addressed to Ulf Reimer: [email protected]
www.jpt.com
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Maksimov et al., (2012) Analysis of clonal type-specific antibody reactions in Toxoplasma
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