Two multiplex assay platforms were used to quantify 23 serologic markers of immune activation and inflammation. The Meso Scale Discovery (MSD, Gaithersburg, MD) system was used to measure the cytokines IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, TNF-α, granular-macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)-γ (Ultra-Sensitive Human Pro-Inflammatory 9-Plex Kit); and chemokine (C-C motif) ligand (CCL)2, CCL4, CCL11, CCL13, CCL17, chemokine (C-X-C motif) ligand (CXCL)10, and IL-8 (Ultra-Sensitive Human Chemokine 7-Plex Kit), according to the manufacturer’s protocols. The MSD platform is a solid-phase electrochemiluminescence-based assay; MSD plates were analyzed on the SECTOR Imager 2400 (MSD, Gaithersburg, MD).
The Luminex platform (Luminex, Austin, TX) was used according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN) to measure soluble (s)CD14, sCD27, sgp130, sIL-2Rα, sIL-6R, sTNF-R2, B-cell activating factor (BAFF), and CXCL13 using a single lot of assay kits, to eliminate lot-to-lot variability. Serum samples were diluted 1:50. The Luminex platform is a fluorescent bead-based assay; Luminex assay data were collected and analyzed using a BioPlex 200 apparatus and BioPlex Manager software (Bio-Rad, Hercules, CA). With both platforms, all samples from an individual were tested on one plate to minimize variability. Each plate contained samples from both HIV-infected and HIV-uninfected men. One additional marker, CRP, was measured by a reference laboratory (Quest Diagnostics) using a high-sensitivity immunonephelometric assay.
The Luminex platform (Luminex, Austin, TX) was used according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN) to measure soluble (s)CD14, sCD27, sgp130, sIL-2Rα, sIL-6R, sTNF-R2, B-cell activating factor (BAFF), and CXCL13 using a single lot of assay kits, to eliminate lot-to-lot variability. Serum samples were diluted 1:50. The Luminex platform is a fluorescent bead-based assay; Luminex assay data were collected and analyzed using a BioPlex 200 apparatus and BioPlex Manager software (Bio-Rad, Hercules, CA). With both platforms, all samples from an individual were tested on one plate to minimize variability. Each plate contained samples from both HIV-infected and HIV-uninfected men. One additional marker, CRP, was measured by a reference laboratory (Quest Diagnostics) using a high-sensitivity immunonephelometric assay.
