Plasma sample preparation and antibody-microarray incubation
The human plasma samples were thawed at room temperature and diluted 5-times in PBS without Ca2+/Mg2+ (pH 6.5) followed by 10,000 g centrifugation in a swing bucket centrifuge for 10 min at Room temperature. The lipid layer on top was carefully removed with the house vacuum. Without disturbing the platelet pellet 300 μl was carefully removed for dialysis (96 well Dispodialyzer/5 kDa, Harvard Apparatus, Holliston, MA) into PBS (pH 6.5) at 4 °C in multiple steps including a last over-night step to yield a maximally pure plasma protein fraction in an appropriate buffer for the biotinylation reaction. The dialyzed plasma was diluted again 6-times in PBS and recombinant Green fluorescent protein (GFP) was spiked into the samples as positive control at a final concentration of 1 μg/ml. The plasma proteins were N-terminally biotinylated (NHS-SulfoBiotin, Thermo Scientific, Rockford, IL), reaction was stopped with 0.1 M glycin final concentration and unbound biotin removed by multiple dialysis against PBS (pH 6.5 and last at pH 8). Then samples were diluted in 3 % casein in PBS (pH 7.4) and the individual samples were incubated on blocked antibody arrays over-night at 4 °C. Blocking was performed by incubating dried arrays in 4 °C precooled 3 % casein in PBS (pH 7.4) overnight on a shaker (30 rpm) at 4 °C. After multiple washing steps antibody-bound protein was detected using 0.5 μg/ml Alexa Fluor 555 conjugated streptavidin (Invitrogen) on a GenePix Pro 4000B scanner (Molecular Devices, Sunnyvale, CA, Additional file 1: Figure S1D). Samples for both AD and svPPA studies were processed in parallel in randomized order in one batch.