Bio plex pro human diabetes assay

Adipokines and peptides were measured as previously described (18 (link)) in all SP and BS samples collected and were measured in triplicate using a magnetic bead-based multiplex assay (Bio-Plex Pro™ Human Diabetes Assay, and Bio-Plex Pro™ Human Hormone Assay, Bio-Rad Laboratories, Inc., USA) according to the manufacturer’s protocol. Data acquisition was performed by a Bio-Plex 200 System™ which uses Luminex fluorescent-bead-based technology (Luminex) to facilitate the analysis of up to 100 different families of color-coded polystyrene beads and allow multiple measurements of the sample ensuing in the effective quantification of adipokines. The Multiplatform luminex assay offers the advantages to use low amount of serum (12.5 to 25 ul of serum per multiplex assay), decreased quality control variability by performing simultaneous assays on a single sample, improved correlated data for multiple analytes, increased sample and finally reduce the manual error during the assay. The working range of the assay is 3,500–280,266 pg/ml with a sensitivity of 0.8 pg/ml and intra-assay CV of 3–4%. Dosage was performed at the Unit of Endocrinology at the University of Rome “Foro Italico”.

As described previously (28 (link)), plasma samples were assayed for glucose and triacylglycerol (TAG) using ILab 650 Clinical Analyser (Instrumentation Laboratory, North Risley, UK). The ILab was calibrated before every experimental run using standard ReferrIL G (Instrumentation Laboratory).
The hormones (insulin, C-peptide, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (total), glucagon, ghrelin, leptin, resistin, visfatin and plasminogen activator inhibitor-1 (PAI-1)) were measured with a multiplex immunoassay (Bio-Plex Pro™ Human Diabetes Assay, Bio-Rad Laboratories Ltd) requiring 50 µL of plasma per sample. The BioPlex software (version 6.1) automatically generated the standard curve using Brendan Scientific's 5-Parametric Logistic Regression. The analyses took place over nine separate runs. All the samples from each participant were measured in the same assay and the intra-assay % CV ranged from 2.5% to 13.2% (n = 3). Missing data from a combination of sources (missing samples, undetectable, outliers) were 5.6%. Each assay plate had at least one person from each of the three study groups.