Detection of fg/mL Levels of IL-2, IL-4, IL-6, IL-10, and IL-17A in Serum, Plasma, and Supernatant of Established Cell Models

Abstract Most immunoassays quantify cytokines with pg/mL sensitivity, which is insufficient to detect those present at low levels. MSD’s next-generation S-PLEXSM assay format was developed to quantify cytokines with fg/mL sensitivity. Here we describe measuring IL-2, IL-4, IL-6, IL-10, and IL-17A levels in normal sera and the supernatants of model cell lines using S-PLEX assays. The assays demonstrated a dynamic range of approximately four orders of magnitude. Standard intra-plate coefficients of variation (CVs) ranged from 3%-15% and inter-plate CVs ranged from 8%–18%. The lower limit of detection (LLOD) was <1 fg/mL for all assays except IL-17A (11 fg/mL). IL-2, IL-6, IL-10, and IL-17A were detectable in 100% of normal sera samples (n=36–75) and IL-4 was detectable in 40% of normal sera samples (n=75). The average concentrations were <1 pg/mL for normal samples. To confirm the sensitivity of these assays and their ability to detect native analytes, we characterized a panel of 23 cell lines that are models for cytokine secretion. As an example, the MOLT-4 cell line derived from acute lymphoblastic leukemia natively expressed detectable levels of IL-2 (1,031 fg/mL), IL-4 (221 fg/mL), IL-10 (60 fg/mL), and IL-17A (30 fg/mL). The expression profiles of these cell lines confirmed the sensitivity of the S-PLEX assays. S-PLEX cytokine assays provide a 100 to 1,000-fold lower LOD than standard ELISAs, allowing the determination of baseline cytokine levels in many sample types.