Microarrays containing synthetic RV-VP1 N terminal peptides, recombinant RSV-G protein and allergens
Comprehensive lists of the chip components spotted on microarrays are reported in Tables S1-2. Synthetic VP1 N terminal peptides representing the three genetic RV species (RV-A: n=18; RV-B: n=9; RV-C: n=10) were selected and produced as described in a previous study (8). The recombinant G protein of the RSV A2 strain was expressed inEscherichia coli BL21(DE3) cells, purified and characterized as described (19). Food allergens used for the calibration of the microarrays were purchased from Sigma Aldrich (Saint Louis, MO, USA) (Table S2). Glass slides containing six microarrays surrounded by an Epoxy frame were obtained from Paul Marienfeld GmbH & Co. KG (Lauda-Königshofen, Germany) and coated with an amine-reactive complex organic polymer, MCP-2 (Lucidant Polymers, Sunnyvale, CA, USA). Microarray components were used at a concentration of 1 or 2 mg/ml in a phosphate buffer (75 mM Na2HPO4), pH = 8.4 and spotted in triplicates using a SciFlexArrayer S12 (Scienion AG, Berlin, Germany). After spotting, printed slides were placed in a humid chamber at 75% relative humidity and incubated at room temperature (RT) overnight. Slides were then blocked with 50 mM ethanolamine in PBS/Tween, pH=9, for 15min and subsequently covered with a plate sealer (Candor, Wangen, Germany). Microarrays were vacuumed and stored at 4°C until use.