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.

Ultrasensitive, Multiplexed Buoyant Sensor for Monitoring Cytokines in Biofluids

“…However, the dynamically secreted concentration of cytokines in biofluids is often below 0.1 pg/mL and therefore not quantifiable. Several recent amplification technologies enabled the detection in the low fg/mL range through plasmon-enhanced fluorescence, 14 biobarcode amplification, 15 photonic-plasmonic coupling, 16 electrochemiluminescence, 17 or molecular on-bead signal amplification. 18 Such low detection limits were achieved with the sample incubation of 1 h or longer and typical sample processing with reagent diluents.…”