Measurement of virus-specific IgG using micro-array technology
For the measurement of virus-specific IgG antibodies, micro-arrays were processed as previously described (13,20). Briefly, micro-arrays were first washed with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (Sigma-Aldrich) for 5 min by stirring and dried by centrifugation (1 min, 1000 g, RT). Serum samples were diluted 1:300 using ImmunoCAP® Specific IgA/IgG Sample Diluent (Phadia, Uppsala, Sweden) and 30 µl of diluted sera, a calibrator and sample diluent for each analysis run were applied onto each microarray and incubated for 2 hours at gentle rocking at room temperature (RT) (Biometra, Jena, Germany). Afterwards slides were washed again, dried by centrifugation and incubated 30 min with 30 µl/per array of affiniPure F(ab’)2 goat anti-huIgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) labelled with DyLight 550 (Pierce, Thermo Fisher Scientific, Rockford, IL, USA). After further rinsing, washing and drying by centrifugation, microarrays were scanned with a confocal PowerScanner (Tecan Grödig, Austria) using 30% of gain (i.e., photomultiplier) and 10% of laser power. Scanned images were analysed using the Mappix software (Innopsys, Carbonne, France).