Sgp130

Fluorescent bead‐based immune‐assays for IL1Ra, IL8, MCP‐1, MIP‐1β, CRP, SAA, MMP1, MMP2, MMP9, sgp130, sICAM1, sVCAM1, sIL2Rα, sIL6R, sTNFRI, sTNFRII, sEGFR, IGFBP1, IGFBP2, IGFBP3, insulin‐like growth factors binding protein‐6 (IGFBP6) and tPAI1 (Millipore Inc.), were used to measure serum levels. Briefly, serum samples were incubated with antibody‐coated microspheres, followed by biotinylated detection antibody. Detection of the proteins was accomplished by incubation with phycoerythrin‐labeled streptavidin. The resultant bead immuno‐complexes were then read on a FLEXMAP3D (Luminex) with the instrument settings recommended by the manufacturer.
The captured median fluorescence intensity (MFI) data was subjected to our quality control steps. Briefly, wells with individual bead counts below 30 or bead count coefficient of variation (CV) above 200 were flagged for exclusion. Replicate wells with CV ≥ 25% were excluded from further analyses. The standard concentration and MFI were log2 transformed before regression. Protein concentrations were estimated using a regression fit to the standard curve with serial dilution of known concentration for each protein.
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Luminex immunoassays for sTNFR-I, sTNFR-II, CRP, SAA, sIL6R, sgp130, sICAM-1, and sVCAM1 were obtained from Millipore (Millipore Inc., Billerica, MA, USA). Multiplex immunoassays were performed according to the manufacturer's instructions. Briefly, serum samples were incubated with antibody-coated microspheres, followed by biotinylated detection antibody. Proteins were detected by incubation with phycoerythrin-labeled streptavidin and the resultant bead immunocomplexes were read on a FLEXMAP3D (Luminex, TX, USA) with the following instrument settings: events/bead: 50, minimum events: 0, flow rate: 60 μL/min, sample size: 50 μL, and discriminator gate: 8000–13500. Median fluorescence intensity (MFI) was collected and used for calculating protein concentration.