Streptavidin conjugated horseradish peroxidase

Anti-CSE1L (H2) antibody-coated 96-well plates (Costar) were blocked with 5% BSA in TBS (Tris-buffered saline) for 1 h. The wells were washed with TBST (0.05% Tween 20 in TBS) and then incubated with serum samples (sixfold dilution with TBS) for 1 h. After being washed with TBST, the wells were incubated with biotin-conjugated anti-phospho-CSE1L antibodies for 1 h. The biotin-conjugated anti-phospho-CSE1L antibodies were prepared by biotinylating anti-phospho-CSE1L antibodies using the Biotin Labeling Kit-NH2 (Dojindo Laboratories, Kumamoto, Japan). The wells were then washed with TBST, reacted with streptavidin-conjugated horseradish peroxidase (R&D Systems, Minneapolis, MN, USA), and followed by incubation with the substrate reagent (R&D Systems). For calibration, two blank wells containing TBST were used as the background value, and two wells that were not coated with anti-phospho-CSE1L antibodies but reacted with all other ELISA reagents were used as control wells. The absorbance at 450 nm was measured within 20 min following the reaction by using a Thermo Multiskan EX microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was assayed two times.

The qualitative analysis of 102 chemokines or cytokines was performed for CSF samples using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems) following the producer's protocol (Table 3). The membranes were incubated overnight with 300 μl of each sample and then washed and incubated with the detection cocktail, with streptavidin-conjugated horseradish peroxidase (R&D Systems) and finally with the chemiluminescence reagent. The spots were visualized by Fusion FX6 (Vilber Lourmat) with EvolutionCapt FX6. The registered signals were proportional to the amount of the bound analyte (Figure 2).

The analysis of 102 chemokines or cytokines was performed on CSF samples using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems) following manufacturers' protocol (Table 3). The kit consists of 4 membranes, which were incubated overnight with 300 μl of every sample and a binding buffer. The membranes were washed and incubated with the detection cocktail, with streptavidin-conjugated horseradish peroxidase (R&D Systems) for 30 min and finally incubation with the chemiluminescence reagent. The spots were visualized by Fusion FX6 (Vilber Lourmat) with EvolutionCapt FX6. The signal readings were proportional to the amount of the bound analyte. The results were analyzed by Zen Software (Zeiss) based on densitometric analysis (Figure 2). The graph bars present means from two spots captured with analyzed antibody according to the following formula: mean density of analyzed spots/mean density of control spots × 100%−mean density of background.

Cell culture supernatants collected at 48h post infection were aliquoted into 200 μL volumes and frozen at -80°C. An ELISA test was conducted for IFN-γ, IL-6 and TNF-α according to the manufacturer’s instructions (R&D Biosystems). Briefly, wells of Nunc Maxisorp 96-well plates were coated with anti-mouse IL-6 (2 μg/ml), anti-mouse IFN-γ (4 μg/ml) or anti-mouse TNF-α (0.8 μg/ml) (R&D BioSystems) overnight at room temperature. Before use, the plates were blocked with PBS containing 1% bovine serum albumin fraction V (BSA, Sigma) for 2 h at room temperature. Cell culture supernatant samples were added to the wells in a volume of 100 μl and the control samples were diluted in PBS 1% BSA. After the 2 h incubation at room temperature, wells were washed 4 times with PBS containing 0.05% Tween 20. The addition of the biotinylated monoclonal antibodies for each of the cytokines (IL-6 150ng/ml; IFN-γ 300ng/ml; TNF-α 50ng/ml) were added and incubated for 2 h at room temperature. Horseradish peroxidase-conjugated streptavidin (R&D Biosystems) was added according to the manufacturer’s recommendations and incubated for 30 min at room temperature. Standard curves were generated using purified recombinant IL-6, IFN–γ and TNF-α according to the manufacturer’s recommendations (R&D BioSystems) using MaxPro generated 4-parameter curve-fit for each cytokine.

Quantification of PMVs in PFP involved use of the home‐made ELISA as described.¹⁶, ¹⁷, ¹⁸, ¹⁹ Briefly, the MVs were captured on the surface of the ELISA plate coated with AD‐1. Then, the PMV detection was achieved by estimating the amount of CD41‐positive MVs. The plate was incubated with biotin‐conjugated mouse anti‐human CD41 monoclonal antibody (Abcam) at 37°C for 2 hour and then with horseradish peroxidase‐conjugated streptavidin (R&D Systems) for 30 minutes. The linear absorbance was read at 450 nm by a microplate reader (Thermo Scientific) after the addition of substrate.