Msd multi spot assay system

The following cytokines were evaluated using the MSD®MULTI-SPOT assay system from MesoScale Discovery, Rockville, MD, USA (www.meso-scale.com): IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, IFN-γ TNF-α [25 (link)]. Peripheral blood plasma was transferred to a 96 well MSD plate and these cytokines were assessed as per manufacturer's instructions. Assays were transferred to five U-PLEX platform plates with calibration curves showing expected signals, sensitivity precision, and accuracy. Sensitivities were < 1 pg/ml for many assays. All assays used the same diluents, diluting linearly from 1–fourfold. Non-specific binding between assays was typically < 0.1%. The assay reproducibility was calculated using the intra variation of the standard curves was shown to be within an acceptable range. The median of the lower limit of detection for all 5 plate runs were as follows: IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, IFN-γ, TNF-α, 0.87, 1.72, 0.84, 0.38, 0.62, 1.15, 17.55, 2.39 respectively, with a closely aligned average value, demonstrating the reproducibility of the assay. Where the sample was below the lower limit of detection the value of the lower limit of detection for that assay was substituted [26 (link)]. No samples reached an upper limit of detection.

Quantities of cytokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, GM-CSF, TNF, and IFN-γ secreted in culture supernatants were measured by MSD® Multi-Spot Assay system (Meso scale diagnostics, MD, USA), according to the manufacturer’s instructions. Sigmoidal samples were analyzed for all 13 cytokines while samples from ascending colon were analyzed for 8 out of 13 cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNF, and IFN-γ).

Supernatants were recovered from monocyte pellets and stored at −80 °C prior to assay. Briefly, thawed supernatants/plasma samples were diluted 1:4 prior to analysis using the MSD® MULTI-SPOT Assay system (Meso Scale Diagnostics, Rockville, MD).

sCD163 was measured in serum and CSF using an in‐house ELISA.³, ²⁴ ELISAs was used to measure CSF levels of total human Tau (h‐Tau), phosphorylated threonine 181‐Tau (p‐Tau), Abeta_1–42 (all by Innotest), and total α‐syn (Analytica Jena Roboscreen GmbH, Leipzig, Germany). MSD MULTI‐SPOT Assay System (Meso Scale Discovery, MD) was used to measure 40 different molecules in serum and CSF (see Supporting Information).

CSF levels of T-tau, P-tau and Aβ42 were quantified with commercially available ELISAs; Innotest® hTau Ag, Innotest® phoshoTau (181P) [26 (link), 27 (link)] and Innotest® β-amyloid 1–42 [28 (link)] (Fujirebio Europe, Gent, Belgium). Aβ1–38 (Aβ38), Aβ1–40 (Aβ40) and Aβ42 were analysed using a MSD Multi-Spot Assay System (Meso Scale Discovery, Rockville, MA, USA) with 6E10 (BioLegend, San Diego, CA, USA) as the detection antibody. The Meso Scale Discovery analyses, which were applied to a subset of patients in the Norwegian cohort, were carried out according to the manufacturers’ procedures.

The level of secreted cytokines in response to BCG was measured with a Meso Scale Discovery (MSD) MULTI‐SPOT Assay System (Meso Scale Discovery, Rockville, MD, USA). Cytokines were analyzed in the supernatants of human primary bladder cancer cultures by the Human ProInflammatory 9‐Plex Ultra‐Sensitive Kit and in mouse MB49 cells by the Mouse ProInflammatory 9‐Plex Ultra‐Sensitive Kit, following instructions from the manufacturer. Controls for standard curves were included with each plate. Data are presented as the means ± standard error of the mean of a minimum of four experiments.