The following biomarkers were measured to assess inflammatory profiles in study participants: C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-a), interleukin-10 (IL-10), monocyte chemoattractant protein 1 (MCP-1), chemerin, omentin, leptin, total adiponectin, high molecular weight (HMW) adiponectin, and fetuin-A. Non-HMW adiponectin was estimated based on the difference between total and HMW adiponectin. Venous blood samples were immediately centrifuged and frozen at −80 °C until analysis. CRP concentrations were determined by a highly sensitive immunoturbidimetric assay using ABX Pentra 400 reagents on an ABX Pentra 400 (Horiba ABX, Montpellier, France). Commercially available ELISA kits were used for the measurements of serum leptin (R&D Systems, Minneapolis, MI, USA), total adiponectin, chemerin, omentin, fetuin-A (all from Biovendor, Germany), and high molecular weight adiponectin (Merck Millipore KGaA, Darmstadt, Germany) and U-Plex assay was used to measure IL-6, TNF-a, MCP-1, and IL-10 (MSD, Rockville, ML, USA). Table S4 provides the detection limits of the kits for the measured biomarkers.
U plex assay
Manufactured by MSD
Sourced in Germany, United States
The U-Plex assay is a multiplex detection platform that allows for the simultaneous measurement of multiple analytes in a single sample. The core function of the U-Plex assay is to provide a flexible and efficient way to analyze complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Mcp 1, by MSD (2 mentions)
Abx pentra 400, by Horiba (2 mentions)
Micro bsa protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rabbit anti α hcg antibody, by Thermo Fisher Scientific (2 mentions)
Interleukin 6 (il 6), by MSD (1 mentions)
Il 10, by MSD (1 mentions)
Fetuin a, by BioVendor (1 mentions)
Omentin, by BioVendor (1 mentions)
Chemerin, by BioVendor (1 mentions)
Triglyceride determination kit, by Merck Group (1 mentions)
A1887, by Merck Group (1 mentions)
A7030, by Merck Group (1 mentions)
Insulin elisa, by Mercodia (1 mentions)
4 protocols using u plex assay
1
Inflammatory Biomarker Assessment in Participants
2
Quantification of hCG and LH in DBS
3
Biochemical Markers and Metabolic Profiling
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Routine markers were measured in serum using ABX Pentra 400 (HORIBA, Kyoto, Japan). Capillary blood glucose concentrations were measured using a glucose oxidase method on a Dr. Müller Super GL (Dr. Müller Glucose Analyzer, Freital, Germany). WISP-1/CCN4 levels were measured by human direct sandwich WISP-1/CCN4 DuoSet ELISA kit (DY1627; R&D Systems, Germany) in combination with bovine serum albumin (A7030, Sigma, Munich, Germany) or human serum albumin (A1887, Sigma) and performed on 96-well high-binding assay plates (82.1581, Sarstedt, Nümbrecht, Germany) as described in [17 (link)]. Commercially available ELISA kits were used for the measurements of serum/plasma insulin (Insulin ELISA, Mercodia AB, Uppsala, Sweden) and TIMP1 (all from R&D Systems, Minneapolis, MN, USA), and the U-Plex assay was used to measure IL6, tumor necrosis factor alpha (TNFα), and MCP1 (MSD, Rockville, MD, USA).
Analysis of hepatic triglyceride content was performed according to the Triglyceride Determination Kit (Sigma Aldrich Chemie, Steinheim, Germany), and the absorbance changes were detected at 540 nm by spectrophotometry.
Analysis of hepatic triglyceride content was performed according to the Triglyceride Determination Kit (Sigma Aldrich Chemie, Steinheim, Germany), and the absorbance changes were detected at 540 nm by spectrophotometry.
4
Quantification of hCG and LH in DBS
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MSD U-Plex assay platform was utilized to assess the hCG and LH values. The U-Plex plates were coated with mouse anti-human hCG beta monoclonal antibody (5H4-E2, Thermofisher) and mouse anti-human LH beta antibody (L1, Abcam), cross-linked with appropriate cross linkers and then washed and incubated with appropriate calibrators as previously described.(16 ) Then the plates were washed with wash buffer 4 times and incubated with the 1st detection antibody of rabbit anti-α HCG Antibody (#R-114-C; Thermo Fisher) for hCG and LH at RT on a shaker at 6500 rpm. Plates were then washed and incubated with 2nd detection antibody of SULFO-TAG™ goat anti-mouse antibody for 30 min at RT on a shaker at 6500 rpm. Plates were washed 4 times and blotted. After the addition of 1x Read buffer (MSD), the plates were read on the MSD 6000 sector imager. All DBS were run in duplicate and both values averaged. For all patients, DBS from both timepoints were run on the same assay. To express analytes concentrations per total mg protein of the DBS extract (as other neonatal DBS studies have done)(28 (link)), total protein of the DBS extracts were measured using Micro BSA protein assay kit (Thermo Fisher, Catalogue #23235).
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