Multiplex immunoassays using Luminex xMAP technology are widely adopted for high-throughput cytokine profiling in small volume biological samples. However, technical artifacts arising from sample components remain underappreciated and can compromise assay interpretation. Here, we discover and investigate a previously unreported Luminex artifact induced by anakinra, a recombinant IL-1 receptor antagonist (IL-1Ra) widely used in clinical and experimental settings. Whole blood or isolated blood immune cells from healthy human volunteers were incubated with different therapeutics/inhibitors/stimulants. Supernatants were harvested for assessment of secreted cytokines using the Human High Sensitivity (HS) Cytokine Premixed Luminex Performance Assay (R&D Systems). MFIs or analyte bead count data was tested for significance using GraphPad Prism, Version 10.4.2. We observed recombinant IL-1Ra to induce drastic elevation of IL-18, IFNα, and IFNγ Luminex readouts when added in vitro to LPS-stimulated human whole blood and isolated blood immune cells. Systematic investigation excluded biological explanations and identified a direct, dose-dependent interaction between exogenous IL-1Ra and the Luminex bead matrix, specifically anti-IL-1Ra beads for measuring IL-1Ra levels alongside other cytokines. This effect, which does not reflect true changes in cytokine secretion, is strictly dependent on the composition of the multiplex assay as it requires the presence of beads for IL-1Ra detection as component of the Luminex kit. IL-1Ra detection bead over-saturation, altered bead counts, and fluorescence spill-over which selectively affect other analytes are likely driving this technical artifact. Our findings highlight a significant and previously unrecognized source of error in multiplex cytokine assays involving a recombinant protein. We stress the importance of rigorous validation and awareness of potential reagent-matrix interactions to ensure robust and interpretable results in immunological studies. • Addition of recombinant IL-1Ra (Anakinra) to whole blood or cell cultures can induce a technical artifact in Luminex assays. • In our Luminex set-up this artifact implies a selective elevation of IL-18, IFNα, and IFNγ expression. • This effect is not induced by biological mechanisms, but depends strictly on presence of anti-IL-1Ra beads in the multiplex panels. • The unexpected recombinant protein interaction with the Luminex matrix highlights the need for thorough multiplex assay validation.
Verweyen et al. (Mon,) studied this question.