Meso scale discovery kit

Serum levels of IL -6 were measured using an electrochemilluminescence method with Meso Scale Discovery kits, and read using the Meso Scale Discovery Sector Imager 2400 (see Richter, 2004 (link) for details regarding this assay technique). Each participant's stored samples were assayed for both IL-6 samples simultaneously, thus allowing thesame controls across both time points for each person. Sensitivity for the IL-6 assayswas 0.3 pg/ml. The intra -assay coefficient of variation (CV) was 1.43% and the inter-assay CV was 4.42%.

Non-fasting blood samples were collected during lab study visits between 7-11 AM to limit diurnal variation. Blood samples provided data on lipopolysaccharide (LPS)-binding protein (LBP) and soluble CD14 (sCD14), two biomarkers produced in response to microbial translocation (Amar et al., 2003 (link); Stehle et al., 2012 (link)). LBP binds LPS and presents LPS to CD14, the receptor for LPS-LBP complexes (Stehle et al., 2012 (link); Ulevitch and Tobias, 1995 (link); Wright et al., 1990 (link)). Samples were measured using an electrochemilluminescence method with Meso Scale Discovery kits, following kit instructions. The stored serum samples for each subject were assayed for each marker in one run, thus using the same controls for both time points. The intra- and inter-assay coefficients of variation (CVs) were LBP were 5.61% and 9.37%, respectively, and the sensitivity was 0.038ng/mL. The intra- and inter-assay CVs for sCD14 were 5.47% and 6.30%, respectively, and the sensitivity was 125 pg/mL.

Fasting blood samples were collected between 7:00 and 9:00 AM to control for diurnal variation. Serum-derived tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were measured by using an electrochemiluminescence method with Meso Scale Discovery kits. Sensitivity was .3 pg/mL, .4 pg/mL, and .2 pg/mL for TNF-α, IL-6, and IL-1β, respectively. The intra-assay and inter-assay CVs for TNF-α were 4.32% and 5.30%, respectively; corresponding values were 1.43% and 4.42% for IL-6 and 4.15% and 4.03% for IL-1β. Each participant’s frozen samples were assayed for all inflammatory markers at once, thus using the same controls for all time points for each person. Noncancer controls and cancer patient samples were mixed on the same plate.

Fasting blood samples were collected between 7:00 and 9:00 AM to control for diurnal variation. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1β (IL-1β) were measured using an electrochemiluminescence method with Meso Scale Discovery kits, and read using the Meso Scale Discovery Sector Imager 2400 (Meso Scale Discovery, Rockville, MD). Sensitivity was .3 pg/mL, .4 pg/mL, and .2 pg/mL for TNF-α, IL-6, and IL-1β, respectively. The intra-assay and inter-assay CVs for TNF-α were 4.32% and 5.30%, respectively; corresponding values were 1.43% and 4.42% for IL-6 and 4.15% and 4.03% for IL-1β. Each women’s frozen samples were assayed for all cytokines in one run using the same controls for all time points for each person. Cytokine data were log transformed to better approximate normality of residuals. A z-score composite of serum cytokines was calculated to obtain a summary measure of inflammation. This method was previously used as a robust representation of overall inflammation (Alfano et al., 2017 (link); Liu et al., 2017 ; Shrout et al., 2020 (link)). The z-score composite demonstrated acceptable levels of internal consistency on average across the three visits (α = .76).