In healthy humans many cytokines are present at levels below the limits of quantification for established multiplex assays. We developed the Simoa Planar Array to enable protein quantification in biological samples with greater sensitivity than traditional ELISAs. In this study we applied Simoa Planar echnology to simultaneous multiplex detection of cytokines, using minimal sample volumes, to achieve sensitivity greater than established multiplex immunoassays. Capture antibodies were printed in a circular pattern in microwells, with biotinylated detector antibodies and chemiluminescent detection used in a sandwich format. Antibody pairs were screened for the ability to quantify analytes with minimal cross-reactivity. Pairs targeting IFNɣ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22 and TNFα were chosen, then assay conditions optimized to minimize matrix interferences.
Analytical validation was performed, and circulating cytokine levels measured in normal human serum and plasma. 12.5 μL of human serum/plasma provided sufficient sample volume per replicate. Sub-picomolar LOD was achieved for all analytes; LLOQs ranged from 0.1 – 1.4 pg/ mL. In normal human serum and plasma, > 80% normal samples were quantifiable (above LLOQ) for most analytes. Analytical validation confirmed sensitivity, precision, specificity and dilution linearity. The 10-plex Simoa Planar assay described here combines ultra- sensitivity with powerful multiplexing, enabling simultaneous detection of 10 cytokines in normal biological samples with superior sensitivity.