Eclia kit

Blood samples were obtained after an overnight fast by venipuncture in vacutainer gel tubes, and serum was separated from cells by centrifugation. Analyses of serum glucose and blood lipids were performed using dry reagent slide technology on the Vitros FS 5.1 (Ortho-Clinical Diagnostics, NY, USA). HbA1c was analyzed by high-performance liquid chromatography (HLPC) using Tosoh HLC-723 G7 (Tosoh Corporation, Tokyo, Japan). Insulin was analyzed by radioimmunossay (RIA) (Millipore Corporation, Billerica, MA, USA) until March 2012, thereafter the electrochemiluminescence immunoassay method (ECLIA) (ECLIA kit, Roche Diagnostics, Mannheim, Germany) was used. Intra- and inter-assay coefficient of variation were < 10% for all assays. Insulin resistance was estimated using the homeostasis model assessment for insulin resistance (HOMA-IR): (insulin (pmol/L) x fasting blood glucose (mmol/L)/135) [33 (link)].

Interleukin-6, 1,25-dihydroxyvitamin D (1,25-OH₂ D), and 25-OH D were analysed from blood taken after an overnight fast and a minimum of 18 hours after the last training session.
Plasma IL-6 was analysed using an ECLIA kit (Roche Diagnostics GmbH, Germany). According to the manufacturer, the limit of detection for this kit was 1.5 pg/mL, and the kit has been found to show no cross-reaction with such substances as IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-8, IFN-γ, and TNF-α. The analyses were performed at the Department of Clinical Biochemistry, Aarhus University Hospital, Denmark.
Serum 25-OH D was analysed using an immunological method that measures both vitamin D 3 and D 2 [18 ]. Analyses were performed at the Department of Clinical Biochemistry, Aarhus University Hospital, Denmark. The lower limit of detection for this analysis was 10 nmol/L. D vitamin status was categorized as being either normal (25-OH D ≥ 50 nmol/L) or deficient (25-OH D < 50 nmol/L) according to the KDIGO guidelines of 2012 [19 ]. Albumin, haemoglobin, C-reactive protein (CRP), and bicarbonate tests were collected from the patients' clinical practice and were analysed in the laboratories of the hospitals comprising those servicing the Capital Region.

The levels of autoantibodies and lipoproteins (HDL-C and LDL-C) in the serum of patients were measured within 1 month of cognitive function testing. Thyroid hormones, including FT3, FT4 and TSH, were measured using a commercially available electrochemiluminescence (ECLIA) kit (Roche). The serum levels of APOE, APOA1, IGF-1 and IGFBP7 were detected using enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s protocols [43 (link)]. The results were fitted to the standard curve, and the detection ranges were as follows: APOE (1.5–400 ng/mL), APOA1 (6.3–200 ng/mL), IGF-1 (62.5–4000 pg/mL), and IGFBP7 (625–40,000 pg/mL).

Samples for biomarker analyses were collected on Cycle 1 Day 1 (predose), Cycle 1 Day 24 (72 h post-dose), and 14 days after the end of treatment. Soluble VEGFR2, VEGF, and CSF1 levels were measured using multiplex enzyme-link immunosorbent assay (ELISA) plates from R&D Systems (Minneapolis, MN), and placental growth factor (PlGF) levels were measured using an electrochemiluminescence immunoassay (ECLIA) kit from Roche Diagnostics (Mannheim, Germany).

All collected samples in March, May, and November 2021 were tested using the Roche Elecsys anti-spike protein of SARS-CoV-2 S, measuring total antibodies [6 –8 (link)]. The Roche assay is an electrochemiluminescent immunoassay (ECLIA) that quantitatively detects antibodies to the SARS-CoV-2 spike protein receptor binding domain (including IgM and IgG) (Roche Diagnostics K.K., Japan). The assay results were interpreted using the manufacturers’ recommended specific thresholds. This assay is highly sensitive and specific [7 (link)]. Assays with good sensitivity have a lower percentage of false negatives. This assay has a measuring range of 0.40–250 U/mL (up to 2500 U/mL with on-board 1:10 dilution), with a concentration of < 0.80 U/mL considered negative and ≥ 0.80 U/mL considered positive.
To compare with an antibody test against spike protein of SARS-CoV-2, we also measured 636 serum samples collected in November 2021 with an anti-nucleocapsid protein of SARS-CoV-2 using an ECLIA kit obtained from Roche diagnostics K.K. (Japan). [10 –14 (link)] According to the manufacturer’s instructions, the cut-off value for a positive SARS-CoV-2 antibody for nucleocapsid protein was deemed as 1 COI (cut-off index).