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.