Meso quickplex sq 120

Cytokine analysis was performed using the Bio-Plex MAGPIX multiplex reader instrument (Bio-Rad, Hercules, CA, USA) and the MesoQuick Plex SQ 120 instrument (Meso Scale Discovery [MSD], Rockville, MD, USA).
1) The Bio-Plex Pro Human cytokine 27-Plex Immunoassay is a 96-well kit that includes all necessary items for the analysis (Cat# M500KCAF0Y; Bio-Rad). The cytokine levels were analyzed according to our previous study [10 (link)11 (link)].
2) Cytokine assays through MSD technology provides a rapid and convenient method for measuring cytokine levels. All vaginal swab samples were analyzed using the MSD multiplex instrument MESO QuickPlex SQ 120 (MSD) [12 (link)]. The whole procedure of analysis for cytokine expressions was explained in our previous study [10 (link)].

IFN-λ4 was detected with a custom-developed electrochemical ELISA on the Meso QuickPlex SQ120 instrument [MesoScale Discovery (MSD)]. Briefly, standard capacity multi-array 96-well plates (MSD) were coated overnight at 4°C with 30 μL of 2 μg/mL custom monoclonal rabbit α-IFN-λ4 antibody. Blocking was performed with bovine serum albumin (MSD blocker A) for 1 h at room temperature with rotation at 400 rpm, followed by 3 washes with PBS and 0.05% Tween. Standards, controls, and experimental samples were prepared using diluent 2 (MSD).
Culture media samples were diluted 1:10 and incubated at room temperature for 2 h with rotation at 400 rpm. After additional wash steps, 25 μL of the 4 μg/mL detection antibody [mouse monoclonal α-IFN-λ4 antibody (buffer exchanged into PBS, MABF227; EMD Millipore)], conjugated with a sulfo-tag (MSD) was added to the wells and incubated for 1 h at room temperature with rotation at 400 rpm. After additional washing, the plates were scanned in a 2× read buffer (MSD) on the Meso QuickPlex SQ120 and the results were analyzed with the MSD Discovery Workbench software. Purified recombinant IFN-λ4 protein (Prokunina-Olsson and others 2013 (link)) was used as a standard at 9 concentrations in the range of 46 pg/mL to 300 ng/mL, and the detection range for the standards was determined as 150 pg/mL to 300 ng/mL.

Biomarker concentrations in serum samples were analysed using the V-PLEX Neuroinflammation Panel 1 Human Kit (Meso Scale Diagnostics (MSD), Gaithersburg, Maryland, USA) on a MESO QuickPlex SQ 120 instrument (MSD) according to the manufacturer′s specifications. Sample values below the detection limit of the assay were assigned a value of the detection limit divided by √2.16 If more than 30% of the measured samples for any given biomarker were below the detection limit, the biomarker was excluded from the analysis. Likewise, samples with a coefficient of variation>30% between duplicate measurements were excluded from the analysis (online supplemental table 1). Biomarkers on the panel included: IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12/IL-23p40, IL-13, IL-15, IL-16, IL-17A, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, TNF-β, eotaxin, eotaxin-3, IFN-γ-induced protein (IP)-10, monocyte chemoattractant protein (MCP)−1, MCP-4, macrophage-derived chemokine (MDC), macrophage inflammatory protein (MIP)-1α, MIP-1β, thymus and activation regulated chemokine (TARC), and CRP.