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.