Quantikine high sensitivity elisa

C‐reactive protein (CRP) and other soluble and cellular biomarkers were measured in blood as part of the baseline collection in the CLSA. C‐reactive protein and albumin were measured in serum using the Cobas 8000 modular analyzer (Roche Diagnostics), TNF and IL‐6 were measured in serum using the Quantikine high sensitivity ELISA (R & D Systems), and absolute measures of monocytes and granulocytes were obtained in whole blood using the Coulter Ac·T diff Analyzer (Beckman Coulter). All measures were log‐transformed and standardized as above.

Multiplex‐assays (Human Magnetic Luminex Performance Assays, R&D systems, Minneapolis, Minnesota) were used for 22/23 serum immune analytes (see Supporting Information), associated in literature with PD/co‐morbidities.³⁹ A sandwich enzyme‐linked immunosorbent assay (ELISA) (Quantikine High Sensitivity ELISA, R&D systems) was used for IL‐17. Exploration in the first 72 participants’ sera (assayed in duplicate) found acceptable intraclass correlations (≥0.9) and left censoring (<10%) for 8/23 analytes: chemokine (C‐C motif) ligand CCL20, epidermal growth factor, IL‐6, IL‐17, leptin, macrophage migration inhibitory factor, resistin and tumour necrosis factor (TNF)‐α.

Serum was collected from clotted peripheral blood collected at each time point by venepuncture into silica containing vacutainers (Becton–Dickinson, Oxford, UK). Serum levels of Interleukin-6 (IL-6), IL-10, monocyte chemotactic protein-1 (MCP-1), brain-derived neurotrophic factor (BDNF) and soluble TNF receptor II (sTNFRII) were measured by Quantikine high sensitivity ELISAs (R&D systems, Minneapolis, USA). Vascular endothelial growth factor (VEGF) was measured using Duoset antibodies (R&D systems, Minneapolis, USA) according to manufacturer’s instructions. Haemoglobin levels were recorded separately as part of a participant’s clinical assessment by the clinical team.

One blood sample was collected per participant into an anticoagulated (EDTA) tube at 8 a.m. on one morning of the study. Samples were centrifuged at 4°C, and aliquots of plasma were prepared for storage within 30 min of collection, and then immediately frozen at −70°C until assays were conducted. At the end of the studies, plasma samples were thawed and assayed for inflammatory markers. Each sample was assayed in duplicate using enzyme-linked immunosorbent assays (ELISAs), according to manufacturer's instructions except as noted. CRP levels were determined by a high sensitivity ELISA (Immundiagnostik, ALPCO Immunoassays, Salem, NH) at a routine sample dilution of either 1:200 or 1:500 (up to 1:2000 as needed), and with an extended standard curve, for an assay range of 0.2–300 mg/L (taking the sample dilution into account). Plasma levels of TNFα and IL-6 were determined by Quantikine high sensitivity ELISAs (R&D Systems, Minneapolis, MN); IL-6 samples were routinely diluted 3-fold, and when necessary, further diluted up to 20-fold in order to measure samples with IL-6 concentrations up to 200 pg/mL (taking sample dilution into account). Measurement of sICAM-1 was performed using a regular sensitivity sICAM-1 ELISA (R&D Systems, Minneapolis, MN). For all four inflammatory markers, intra-assay variability was less than 6% and inter-assay variability was less than 8%.