Multisite Comparison of High-Sensitivity Multiplex Cytokine Assays

Abstract

The concentrations of cytokines in human serum and plasma can provide valuable information about in vivo immune status, but low concentrations often require high-sensitivity assays to permit detection. The recent development of multiplex assays, which can measure multiple cytokines in one small sample, holds great promise, especially for studies in which limited volumes of stored serum or plasma are available. Four high-sensitivity cytokine multiplex assays on a Luminex (Bio-Rad, BioSource, Linco) or electrochemiluminescence (Meso Scale Discovery) platform were evaluated for their ability to detect circulating concentrations of 13 cytokines, as well as for laboratory and lot variability. Assays were performed in six different laboratories utilizing archived serum from HIV-uninfected and -infected subjects from the Multicenter AIDS Cohort Study (MACS) and the Women's Interagency HIV Study (WIHS) and commercial plasma samples spanning initial HIV viremia. In a majority of serum samples, interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha were detectable with at least three kits, while IL-1␤ was clearly detected with only one kit. No single multiplex panel detected all cytokines, and there were highly significant differences (P < 0.001) between laboratories and/or lots with all kits. Nevertheless, the kits generally detected similar patterns of cytokine perturbation during primary HIV viremia. This multisite comparison suggests that current multiplex assays vary in their ability to measure serum and/or plasma concentrations of cytokines and may not be sufficiently reproducible for repeated determinations over a long-term study or in multiple laboratories but may be useful for longitudinal studies in which relative, rather than absolute, changes in cytokines are important.

How to: Measuring blood cytokines in biological psychiatry using commercially available multiplex immunoassays

“…The sensitivity was generally higher for the V-PLEX® platform, compared to Luminex® platform, as previously reported (Breen et al, 2011;Chowdhury et al, 2009). Despite the large concentration range obtained for the Luminex® standard curves, the slope steepness was higher with V-PLEX® for the low concentrations, and a better conservation of linearity was achieved (Supplementary Figure 1, Table 1).…”

“…Indeed, both platforms failed to robustly detect the GM-CSF, IL-1β, IL-2 and IL-5 cytokines for which previous investigations had reported significant variations in expression either during MDE treatment or at baseline in comparison to healthy controls (Biancotto et al, 2013;Dahl et al, 2014;Hernandez et al, 2008;Schmidt et al, 2014;Shelton et al, 2015). These types of inconsistency in the results have already been reported (Breen et al, 2011;Chaturvedi et al, 2011;Dabitao et al, 2011;Dupuy et al, 2013;Fu et al, 2010). In particular, the consensus that the detection of IL-1β suffers from poor performances is of importance.…”

“…Thus far, very few investigators have compared these technologies and evaluated the limits of quantification and intra-assay reproducibility for assay of human blood (Breen et al, 2011;Chowdhury et al, 2009;Fu et al, 2010;Nechansky et al, 2008;Toedter et al, 2008).…”

Simple Plex™: A Novel Multi‐Analyte, Automated Microfluidic Immunoassay Platform for the Detection of Human and Mouse Cytokines and Chemokines

“…Consequently, multiplexing capability is becoming a critical parameter of biomarker validation and testing as biomarkers are increasingly utilized in all facets of life science research and clinical diagnostics. The adoption of multiplexed testing formats in either clinical research or diagnostics has been severely limited for many reasons, including technical concerns regarding assay reproducibility, cross‐reactivity between the disparate antibody assay components, decreased sensitivity, increased variability, and reported non‐correlation of results with existing methods, including single‐parametric ELISAs 6, 7. As such, many potential multiplex tests continue to be performed as single‐test ELISAs in a parallel processing format.…”

Soluble Intercellular Adhesion Molecule-1: A Potential Biomarker for Pain Intensity in Chronic Pain Patients

“…Serum levels of 30 pro-and anti-inflammatory cytokines were measured by Myriad Rules-Based Medicine, (Texas, USA), using the Human Cytokine multiplex immunoassay (Human Cytokine Map A+B). The multiplex microbead assay is based on Luminex technology and measures proteins in a similar manner to standard sandwich ELISA, with comparable sensitivity and range [18,19]. Measurements below the limit of detection were set to 50% of the detection limit.…”