V plex assay

Plasma was thawed on ice and TNFα, IL-6, IFNγ, and IL-1β were quantified by the U-PLEX Assay and CRP was quantified using the V-PLEX Assay according to manufacturer’s instructions (Meso Scale Discovery, Rockville, MD). Both assays were evaluated on the QuickPlex SQ 120 Instrument (Meso Scale Discovery).

Serum samples from all participants were collected from their blood. After thawing, serum samples were centrifuged for 3 minutes at 2000 g to remove particulates prior to sample preparation and analysis. The electrochemiluminescence V-PLEX assay (Meso Scale Discovery, MD) was used to measure proinflammatory proteins (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-a), cytokines (IL-15, IL16 and VEGF) and chemokine (CXCL10). Serum samples were diluted 2-fold and measured in duplicate. The cytokines concentration was determined with the electrochemiluminescent labels whilst the plate is inserted into the MSD instrument (MESO QUICKPLEX SQ 120). All samples were assayed in duplicate. High and low controls were used to assess variance between plates. The inter-assay coefficient of variations was <10%. The results were analyzed using MSD DISCOVERY WORKBENCH analysis software.

The majority of the complement proteins in this study were measured using customised v-plex assays from MesoScale Discovery (MSD; Rockville, Maryland, USA), using antibody pairs developed in-house. C3, C4, factor B (FB), factor H (FH) and factor I (FI) were analysed together in multiplex 1 and factor D (FD), Bb, C3a, iC3b and terminal complement complex (TCC) were analysed together in multiplex 2. All 8 analytes were measured by an electrochemiluminescence (ECL) immunoassay technique according to the manufacturer’s protocol. In brief, pre-coated plates were blocked with 150 μl/well 3% BSA in PBS at room temperature for 2 h with shaking at 600 rpm. Plasma samples were diluted 1:2000 for multiplex 1 and 1:2 for multiplex 2 in assay buffer (PBS containing 1% BSA and 10 mM EDTA) and 25 μl aliquots were added in duplicate to wells. A calibration curve comprising a series of fivefold dilutions of protein standard was run in duplicate on each plate. Plates were incubated with shaking at 600 rpm at room temperature for 60 min. After washing in PBS containing 0.01% Tween20, 25 μl of a mixture of the relevant SULFO-TAG-labelled detection antibodies diluted in assay buffer (1:100) was added and incubated with shaking at 600 rpm at room temperature for 60 min. After washing, 150 μl of 2 × MSD reading buffer was added to each well and ECL signal was measured on the Sector Imager 2400 (MSD).

BUN was measured using the urease indophenol method (Wako Pure Chemical Industries). An absorbance 96-well plate reader (Spectra MAX Plus; Molecular Devices, Sunnyvale, CA, USA) was used at a wavelength of 570 nm. The concentrations of IL-1β, IL-2, IL-6, TNF-α, and IL-10 were determined in the plasma, using custom V-plex assays (Meso scale discovery, Rockville, MD, USA) according to the manufacturer’s protocol. The plasma HMGB1 concentration was measured using an established enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (Fuso pharmaceutical, Osaka, Japan).

IL1-α, IL-8, and VEGF-A in cell supernatant were analyzed using Quantikine ELISA kits (R&D Systems; DLA50, D8000C, DVE00). Human GRO-beta and GRO-gamma ELISA Construction kits (Antigenix America Inc.; RHF810CK, RHF820CKP) were used to measure the supernatant and plasma levels of CXCL2 and CXCL3, respectively. IL-1α, IL-8, and VEGF-A in human plasma were analyzed using custom V-PLEX Assays (Meso Scale Discovery, Maryland, USA; K151RBD-2, K151RAG-1, K151RHD-1). Patient blood samples were obtained after multicenter ethics approval (09/H1102/107 and 14/YH/0090) as described previously [16 (link), 22 (link)]. Blood was centrifuged within 1 h of collection at 2500 rpm in a lithium-heparin containing tube; the plasma was then removed, aliquoted, and stored in a − 70 °C freezer prior to thawing for ELISA analysis. ELISAs were carried out according to the manufacturer’s instructions.