eBook: Multiplex Technology Guide - 11

LMX detected many cytokines in actual plasma
samples that were entirely missed by MSD.

Bead-based LMX system quantified 13/16
analytes at higher levels than MSD.

LMX total assay time was 5% lower, and labor
time was 36% lower than with MSD.

quantification (ULoQs) were higher in LMX vs. MSD
assays for 13/16 shared analytes. Surprisingly, while
MSD calibration curves indicated lower LLoQs for
most shared analytes, many more cytokines in plasma samples went undetected by MSD than by LMX.
Bland-Altman plots indicated that the MSD platform
classified 13/16 shared analytes at lower concentrations than the LMX system. Total assay times averaged 20 hours and 28 minutes and 21 hours and 33
minutes for LMX and MSD, respectively, including an
overnight incubation. Labor times averaged 1 hour
and 37 minutes for LMX and 2 hours and 33 minutes
for MSD. While MSD standard and QC results promised better cytokine detection sensitivity, LMX markedly outperformed MSD when assessing actual human plasma samples.

Luminex's xMAP Technology showed significantly better real-life performance than the MSD system for rapidly quantifying multiple cytokines in
small-volume human plasma samples.

Abstract
Multiplex immunoassays quickly and simultaneously quantify multiple analytes in biological samples-
up to 500 discrete biomarkers with Luminex (LMX)
technology. Multiplexing technology offers the ability to concurrently evaluate numerous cytokines and
biomarkers, providing time-, labor-, and resourcesaving advantages vs. conventional assays that only
measure one analyte at a time. Because our core
laboratory performs multiplex immunoassays using
both the bead-based fluorescence MILLIPLEX® assay with Luminex xMAP® Technology and the Meso
Scale Discovery (MSD) planar electrochemiluminescence system, we compared their performance in
the 96-well microplate format. We measured cytokines in standards, quality controls (QCs), and human
plasma samples (n=62), and recorded assay time
requirements for both systems. Achieving desired
analyte overlap with a 21-plex LMX assay required
using three 10-plex MSD kits, yielding 16 common
analytes. Both assays provided good reproducibility in all standard curves. Maximum inter-assay CVs
of shared analytes with kit QCs were 18.2% for LMX
and 13.9% for MSD. The MSD platform had lower
limits of quantification (LLoQs) than the LMX system for 14/16 shared cytokines. The upper limits of

Introduction
Cytokines and chemokines (chemotactic cytokines)
constitute a growing group of diverse, small (<40
kDa) secreted bioactive protein molecules that are
essential in cross-communication between cells and
tissues.1,2 The simultaneous but balanced release
of multiple cytokines is important for maintaining
normal homeostasis, and is also an essential component of a well-regulated immune response following
pathogen exposure, trauma, and in disease states.
Multiplex immunological assays employ a variety of
technologies, including planar chemiluminescence
and bead-based immunocapture platforms, to simultaneously evaluate levels of multiple circulating
proteins, including cytokines and other biomarkers.3
These multiplex immunoassays help to rapidly identify complex cytokine expression signatures that
occur in diverse disease states, can enhance and expand epidemiological research, and are emerging
as valuable clinical diagnostic and prognostic tools.
This utility is supported by the steadily increasing
number of multiplex proteomic assays that receive

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