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).