Background: Many diseases result in prolonged and inappropriate inflammatory responses that are characteristic of the underlying pathology. Novel methods to detect cytokines important in immune regulation and oncology may enable early detection of these diseases and lead to favorable treatment outcomes. A six-plex panel including IL-6, IL-10, IL-12p70, IL-17A, TNFa and IFNg was evaluated for sensitivity, linearity, spike/recovery and cross reactivity.
Methods: A fully automated multiplexed ultra-sensitive digital immunoassay for inflammatory biomarkers was developed based on Single Molecule Array (Simoa) technology. The assay reagents were designed for use on the Simoa platform. In the first step, an immunocomplex was formed by incubating calibrators or samples with capture antibody-coated paramagnetic beads and biotinylated detector antibody. Then the immunocomplex was labeled with streptavidin-β-galactosidase conjugate followed by incubation with the fluorogenic substrate resorufin-β-D-galactopyranoside. Beads were loaded into a microwell array to produce single beaded wells and digital signals. The individual assays in the plex were evaluated for sensitivity, linearity, spike/recovery, cross reactivity and detectability in serum and plasma from 20 healthy donors.
Results: The limits of detection were 0.0073 pg/mL (IL-6), 0.0056 pg/mL (IL-10), 0.0094 pg/mL (IL-12p70), 0.0127 pg/mL (IL-17A), 0.0077 pg/mL (TNFa) and 0.0126 pg/ml (TNFa). LODs were comparable between multi- and single-plex assays. Spike/recovery and dilution linearity were all within 74-101%. There was minimal cross reactivity between individual assays. Analysis of plasma and serum samples from healthy donors showed all six analytes were readily detected with levels averaging in the low pg/ml to sub pg/ml.
Conclusions: The Simoa human 6-plex assay is able to reliably measure inflammatory biomarkers simultaneously in plasma and serum samples from healthy individuals. It provides a new tool for the quantification of IL-6, IL-10, IL-12p70, IL-17A, TNFa and IFNg in both normal and disease samples, facilitating the investigation of inflammation and Immuno-Oncology applications.