Multiplex elisa

For the subset of the original population that underwent subsequent cardiometabolic testing (n = 42), our health care provider team performed a physical examination on each of these patients and obtained each patient’s demographic data, clinical history, and anthropometric measurements. Blood samples were collected after an overnight fast and analyzed for basic chemistry, complete lipid profile, and high sensitivity C-reactive protein at the NIH Clinical Center. Interleukins (IL)-1β, IL-6, IL-18 were measured using a multiplex ELISA (Meso Scale Diagnostics, Rockville MD, USA), as described previously (Bastarache, Koyama, Wickersham, & Ware, 2014 (link)).

Plasma Hp concentrations by routine clinical chemistry used immunoturbidimetry at 604 nm absorbance on an Abbott Architect analyzer. The measurement interval for this assay was 0.1–2.7 g/L. Samples with concentrations above this were diluted and re-run. Reference interval (> 17 years of age) was 0.24–1.9 g/L. No reference data was available for those < 17 years of age. Healthy volunteers in this study recruited from the local population therefore provided reference data. CRP and SAA (liver derived positive APPs) were measured by multiplex ELISA (Meso Scale Diagnostics, MA, USA). The clinical chemistry unit assayed CRP (in some samples to bridge CRP values from the multiplex kit), albumin (negative APP) and ESR (to calculate JADAS27).

Cells (2 × 10⁴ per well) were cultured for 48 h and the supernatants harvested. Matrix Metalloproteinase 3 (MMP3) and Tissue Inhibitor of Metalloproteinase 3 (TIMP3) were measured by single-plex ELISA (R&D Systems, Minneapolis, MN, USA), IP10, monocyte chemoattractant protein 1 (MCP1) and macrophage inflammatory protein 1 alpha (MIP1α) were measured via multiplex ELISA (Meso-scale discovery, Rockville, MD, USA).

Cervico-vaginal secretions (CVS) collected by SoftCup as described previously (see method) were diluted 10-fold using sterile PBS and spun down at 1730g for 10 min. Subsequently, the supernatant was frozen at −80 °C for cytokine analysis. The levels of cytokines IL-1α, IP-10, IL-8, MIP-3α, MIP-1β, IL-17a, IFN-α2a, IL-6, and MIG were measured in duplicate by Multiplex ELISA according to the protocol (Meso Scale Discovery, Rockville, MD). The supernatant of cervical secretion was plated at 25 μl per well. The standard curve was used to determine the lower and upper limit of detection and concentration of each analyte (pg/ml). Any sample above the upper level of detection was diluted and the Multiplex ELISA was repeated. The LLODs were as follow: IFNα2a = 4.82 pg/ml; IL-17 = 8.3 pg/ml; MIP-3α= 2.7 pg/ml; IL-6 = 1.27 pg/ml; IL-1α= 99.6 pg/ml; IL-8 = 1.24 pg/ml; MIG = 2.47 pg/ml; IP-10 = 42.1 pg/ml; MIP-1β= 11.5 pg/ml. Samples that were below the limit of detection (LLOD) were given the LLOD value. Samples that were above the LLOD with a CV repeatedly higher than 30 were excluded from analysis. All samples were run by a researcher blinded to the status of participants. CVS samples provided at both study visits were assessed on the same plate to account for the plate-to-plate variability.