Bep 2000 elisa analyser

Blood samples drawn at admission included liver enzymes, coagulation factors, hematology, fluid balance, kidney function measures, and serum zinc. We used standard methods at the local Department of Clinical Biochemistry. We calculated the MELD score to show patients characteristics for all WD patients even in the absence of end stage liver disease. The cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-8 were analyzed in doublets using a multiplex cytokine assay (V-PLEX Proinflammatory Panel 1 (human), MSD Rockville, USA).
Measurements of P-sCD163 were performed in doublets by a validated in-house sandwich enzyme-linked immunosorbent assays in plasma using a BEP-2000 ELISA-analyser (Dade Behring), as previously described; sCD163 is stable for years at − 80 °C and when exposed to repeated freeze and thaw cycles [17 (link)]. Control samples were included in each run.

Plasma samples were analyzed using commercially available kits according to the manufacturer’s instructions. High sensitivity C-reactive protein (hsCRP) was measured on a Siemens STRATUS CS in duplicate with a coefficient of variance (CV) of 8.6%.
Soluble CD163 (sCD163) was measured by an in-house sandwich enzyme-linked immunosorbent assay using a BEP-2000 ELISA-analyser (Dade Behring, Deerfield, IL, USA).16 (link)
Soluble endothelial selectin (sE-selectin), soluble vascular cell adhesion molecule1 (sVCAM-1), soluble intercellular adhesion molecule 1 (sICAM-1), matrix metalloprotease 9 (MMP-9), and total plasminogen activator inhibitor 1 (PAI-1) were analyzed on a multiplex assay (Milipore Corporation, MA, USA) using Luminex^® technology.
All samples were run in duplicates with CV < 12%. CD4 cell counts and HIV RNA levels were done routinely on blood and plasma when collected. Serum lipids were analyzed on a MODULAR ISE 1800 (Roche, Basel, Switzerland).
Metabolic syndrome was defined as having at least two of the following: fasting blood glucose ≥ 5.6 mmol·L⁻¹ or (systolic blood pressure ≥ 135 mm Hg or diastolic blood pressure ≥ 85 mm Hg) or (triglycerides > 1.7 mmol·L⁻¹ or high density lipoprotein <1.03 mmol·L⁻¹).17 (link)
Framingham risk score (FRS) was calculated as the 10-year risk of coronary heart disease (CHD) according to published definitions.18 (link)

HbA1c, ALAT, triglyceride, and cholesterol concentrations were measured using standard clinical biochemical methods. Plasma glucose concentrations were measured using an YSI 2300 STAT Plus glucose analyzer (YSI, Inc., Yellow Springs, OH, USA). Serum insulin concentrations were measured using an immunoassay kit (Dako, Inc., Glostrup, Denmark). We determined serum sCD163 concentrations in duplicate samples that had been frozen for up to 3 years at −20 °C by use of an in-house sandwich ELISA on a BEP-2000 ELISA analyser (Dade Behring, Inc., Marburg, Germany) (32) (link). In each run of 36 samples, we co-analyzed control samples and serum calibrator with concentrations traceable to purified CD163. The interassay imprecision in the current studies (seven runs) was 3.0% coefficient of variation (CV) at a concentration of 1.28 mg/l, and 7.5% CV at 3.37 mg/l. The limit of detection was 6.25 μg/l. sCD163 is robust to thawing, and stability has been rigorously verified for at least 7 months at −20 °C (32) (link).

Samples for the determination of sCD163 and sMR were drawn in 9 ml EDTA tubes. The tubes were centrifuged at 4°C for 10 min at 3000 rpm. The plasma was then removed, aliquoted and stored at -80°C until analysis.
The levels of serum sCD163 and sMR were analysed in duplicate using samples of frozen serum with an in-house sandwich ELISA on a BEP2000 ELISA analyser (Dade Behring, Marburg, Germany) as previously described [21] (link) (submitted to Journal Of Leukocyte Biology). Control samples were included in each run. The inter-assay imprecision in the current study for sCD163 was 1.9 CV% and 5.2 CV% at levels of 1.91 mg/l and 3.52 mg/l, respectively. The sMR imprecision was 3.5 CV% and 4.2 CV% at levels of 0.51 mg/l and 1.13 mg/l, respectively. Both sCD163 and sMR are resistant to thawing degradation, and their stability has been verified at −80°C for at least 12 months.