2.10 | Epitope mapping using peptide array
Peptide microarrays of 15-residues cyclic peptides, derived from the
EspB sequence and containing an overlap of 11 residues, were obtained
from JPT Peptide Technologies GmbH. Each microarray included three
identical subarrays as technical triplicates. Full-length EspB protein
was spotted on the array and used as a positive control, while bovine
serum albumin (BSA) served as a negative control. The binding of
mAb-EspB-B7 to the peptide array was carried out according to the
manufacturer’s instructions (www.jpt.com), with minor modifications.
Briefly, 20 μg/mL mAb-EspB-B7 (0.1% TBST v/v) were incubated on the
peptide microarray for 2 hr at room temperature. The peptide microarray
slides were then washed (five times with TBST), incubated with Alexa
Fluor 647-affinipure mouse anti-human IgG (Jackson ImmunoResearch) for
45 min at room temperature, washed (five times with TBST and then five
times with doubly distilled H2O), and dried.
Fluorescence was detected with a GenePix 4000B scanner (Molecular
Devices) at a resolution of 10 μm pixel size and analyzed by the Genepix
Pro 6.0 analysis software (Molecular Devices). Signals were normalized
and plotted to reflect the relative intensities of the fluorescence
signals.