Milliplex assay kits

Plasma samples were collected twice in pairs from all patients pre‐ and post‐treatment. Overall, 17 plasma angiogenesis‐related mediators (hepatocyte growth factor [HGF], placental growth factor [PlGF], VEGF‐A, VEGF‐D, angiopoietin‐2, interferon‐γ, interleukin‐6 [IL‐6], IL‐8, soluble neuropilin‐1, thrombospondin‐2 [TSP‐2], osteopontin [OPN], soluble vascular endothelial growth factor‐receptor‐1 [sVEGFR1], sVEGFR2, sVEGFR3, soluble intercellular adhesion molecule‐1 [sICAM‐1], soluble vascular cell adhesion molecule‐1 [sVCAM‐1], and tissue inhibitor of metalloproteinase‐1 [TIMP‐1]) were analyzed using the multiplex assay using Luminex® technology.
First, angiogenesis factors from plasma samples were bound to their respective specific antibodies using Luminex® beads. Next, biotinylated detection antibodies were conjugated to antigen–antibody complex and labeled with streptavidin‐phycoerythrin. Subsequently, beads were analyzed using Luminex® 200, and fluorescent signals were calculated as concentrations from the standard curve for each substance. Finally, these angiogenesis factors were measured using Milliplex® assay kits (Merck).¹³, ¹⁴

Plasma samples were collected twice in pairs from all patients pre‐ and post‐treatment. Overall, 17 plasma angiogenesis‐related mediators (hepatocyte growth factor [HGF], placental growth factor [PlGF], VEGF‐A, VEGF‐D, angiopoietin‐2, interferon‐γ, interleukin‐6 [IL‐6], IL‐8, soluble neuropilin‐1, thrombospondin‐2 [TSP‐2], osteopontin [OPN], soluble vascular endothelial growth factor‐receptor‐1 [sVEGFR1], sVEGFR2, sVEGFR3, soluble intercellular adhesion molecule‐1 [sICAM‐1], soluble vascular cell adhesion molecule‐1 [sVCAM‐1], and tissue inhibitor of metalloproteinase‐1 [TIMP‐1]) were analyzed using the multiplex assay using Luminex® technology.
First, angiogenesis factors from plasma samples were bound to their respective specific antibodies using Luminex® beads. Next, biotinylated detection antibodies were conjugated to antigen–antibody complex and labeled with streptavidin‐phycoerythrin. Subsequently, beads were analyzed using Luminex® 200, and fluorescent signals were calculated as concentrations from the standard curve for each substance. Finally, these angiogenesis factors were measured using Milliplex® assay kits (Merck).¹³, ¹⁴

On the same day of clinical and ultrasound assessment, we obtained and stored the patients’ sera at –80°C. On the basis of previous reports [30 (link), 37 (link), 38 (link)], we measured serum levels of β-defensin 2 and calprotectin using ELISA kits (Arigo, Hsinchu, Taiwan; Hycult Biotech, Uden, The Netherlands, respectively). We also measured serum levels of CCL20/MIP3a, GM-CSF, IFN-γ, IL-1β, IL-6, IL-8, IL-9, IL-10, IL-12p70, IL-15, IL-17A, IL-17F, IL-21, IL-22, IL-23, Lipocalin-2/NGAL, and TNF-α using MILLIPLEX Assay Kits and a MAGPIX Multiplexing System (Merck Millipore, Darmstadt, Germany). Data were analyzed using xPONENT 4.2 Software (Luminex Corporation, Austin, TX, USA) and the average of duplicate samples was calculated.
One RA patient with a result of extremely high values for most of the cytokines/chemokines was excluded from cytokine/chemokine analyses as an outlier due to measurement error.