Sample analysis
The samples were analysed using an in-house developed multiplex assay
based on Meso-Scale Discovery (MSD) technolog, targeting 3 biomarkers
for inflammation (Interleukin-18 (IL-18), Monocyte Chemotactic Protein
(MCP-1), C-Reactive Protein (CRP)), 1 anti-inflammatory marker (soluble
Tumor Necrosis Factor 1 (sTNF RI)), 1 biomarker for stress (Heat Shock
Protein-70 (HSP70)), 2 growth factors (Epidermal Growth Factor (EGF),
Vascular Endothelial Growth Factor A (VEGF-A)), and 3 neurotrophic
biomarkers for brain development and/or damage (Brain-Derived
Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), and S100 calcium
binding protein B (S100B)).
All antibodies were purchased from RnDsystems, with the exception of
anti-S100B, which was purchased from Sigma-Aldrich (capture) and DAKO
(detection), respectively. The capture antibodies were purchased
biotinylated, except for anti-S100B that was in-house purified using
protein A column, and biotinylated using EZ-Link Sulfo-NHS-LC-Biotin
(ThermoFisher) as per manufacturers instructions. The biotinylated
antibodies were diluted to the concentration 0.1µg/ml for CRP and
10µg/ml for the other antibodies, bound to different linkers 1-10 (MSD)
according to manufacturer’s instructions, and finally added to each
plate well (50µl/well, U-plex plates, MSD), and incubated at room
temperature (RT) for 1 hour. After washing with washing buffer (PBS
containing 0.05% Tween 20), the plates were stored at 4°C until use.
Calibrators and controls were prepared by recombinant antigens diluted
in diluent 7 (MSD). All detection antibodies were sulfo-tagged using MSD
Gold Sulfo-tag NHS-Ester (MSD) as per manufacturers instructions, and
were mixed together to a final concentration of 0.1µg/ml of each
antibody.
The DBSS were punched as 2x3.2mm disks in microtiter wells using DBS
Puncher® instrument (Perkin Elmer), added 130 µl extraction buffer (PBS
containing proteaseinhibitor, as earlier described (27)) to each well,
sealed and extracted for 1 hour at RT on a shaker set at 450 rpm. After
extraction, 50µl extract was aspirated from each well and transferred to
the pre-coated plates with a pipetting robot (Biomek 4000, Ramcon)
together with calibrators and controls. The plates were sealed and
incubated on a shaker set at 600 rpm for 2 hours at RT. After washing,
detection antibodies were added to each well followed by 2 hours
incubation at 600 rpm at RT.
Finally, the plates were washed, added 2xRead buffer T (MSD), and
immediately read at the QuickPlex reader (MSD).
Concentrations of the biomarkers were calculated from the calibration
curves with 4 parameter logistic fit using Discovery Workbench 4.0
software (MSD).
Analytical characteristics are shown in table 2. Values measured below
or above the working ranges were set to the lowest or highest measurable
concentration.