2.4. Analysis of the Specificity of Recognition of Monoclonal Igs Using PEPperCHIP® Infectious Epitope Micro-Arrays

We used the commercial micro-arrays called PEPperCHIP^® Infectious Disease Epitope microarrays, from PEPperPRINT Gmbh (Heidelberg, Germany), which cover 3760 linear B-cell epitopes of 196 different pathogens associated with infectious diseases of the immune Epitope Database. The 3760 linear B-cell epitopes were translated into 4344 different peptides printed in duplicate (8688 peptide spots) and framed by poliovirus (KEVPALTAVETGAT, 42 spots), c-myc (EQKLISEEDL, 30 spots) and HA (YPYDVPDYAG, 40 spots) control peptides. Microarrays were placed in suited PEPperCHIP^® incubation trays (PEPperPRINT GmbH, Heidelberg, Germany). After 15 min pre-swelling in washing buffer and 30 min in blocking buffer MB-070 (Rockland, Gilbertsville, PA, USA), one PEPperCHIP^® Infectious Disease Epitope Microarray was incubated with the secondary goat anti-human IgG (Fc) DyLight680 antibody (1:5000) (Rockland, Gilbertsville, PA, USA) and control mouse monoclonal anti-HA (12CA5) DyLight800 antibody (1:2000) for 45 min at room temperature to analyze background interactions with the infectious disease epitopes. Subsequent incubation of PEPperCHIP^® Infectious Disease Epitope Microarrays with the purified monoclonal IgG (50 or 100 µg/mL) of patients was followed by staining with secondary and control antibodies for 30 min at room temperature and 200 rpm orbital shaking. The peptide microarrays were washed 3 × 1 min with PBST and rinsed with deionized water. After drying in a stream of air, images were recorded using an Odyssey Imaging System (LI-COR, Lincoln, NE, USA) at a wavelength of 700 and 800 nm with a resolution of 21 μm and a scanning sensitivity of for each channel 7. The HA control peptides were simultaneously stained as internal quality control to confirm the assay quality and the peptide microarray integrity. Image analysis and quantification of array data were done with PepSlide^® Analyzer (Sicasys Software GmbH, Heidelberg, Germany). These experiments were performed and results of the binding of purified monoclonal Igs were provided by the PEPperCHIP^® microarray manufacturer (PEPperPRINT).