ELISA and Multiplex Immunoassays
Olink multiplex assaySerum samples were analyzed with the Olink Inflammation I Proseek multiplex assay (95302) according to manufacturer’s specifications. The levels of analyte-specific DNA amplicons for each patient were quantified by Fluidigm Biomark HD.
Luminex bead-based multiplex immunoassayLuminex bead-based sandwich immunoassay was used to assess serum protein levels quantitatively and simultaneously on the Millipore Luminex 200 Bead Reader System. Two MILLIPLEX MAP Human Cytokine/Chemokine 41-plex Magnetic Bead Panels (HCYTOMAG-60K-PX41 and HCP2MAG-62K-PX23) were assayed on Curiox Biosystems DA-Bead plates and plates were washed on a DropArray LT210 washing station following the Curiox low-volume protocol.
ELISAFor additional quantitative assessment of important analytes conventional sandwich enzyme-linked immunoassays (ELISA) were utilized including Periostin (DY3548B) and sST2 (DST200) from R&D Systems. Levels of IL-22 and TARC were measured using MesoScale Discovery (MSD)-based sandwich ELISA. The IL-22 assay, consisting of proprietary antibodies and recombinant protein, utilizes a Small Spot SA–MSD plate (L45SA-1) and in-house diluents. Following incubations, wells were washed with 1x TBST using a Bio-TEK ELx405 and analyzed on the Meso Quick Plex SQ 120 plate reader. TARC (K151NTD) was measured according to manufacturer’s specifications. Levels of IL-13 (102732) and IL-17A (101599) were assessed by Quanterix single molecule array (Simoa) bead-based 2.0 assays on the Simoa HD-1 analyzer per manufacturer protocols.