eBook: Multiplex Technology Guide - 13

were comprised of 20 healthy individuals, 21 diagnosed with diabetes mellitus, and 21 individuals
with elevated PCT levels (>3 ng/mL)-an indicator
of sepsis.12,17,18 Samples were purchased from Central
BioHub GmbH (Hennigsdorf, Germany), stored at
-80°C until use, and only underwent a single thawing event. Study performance complied with the
Declaration of Helsinki. Demographic information
on sample donors is provided in Table 1.

Methods
The complete published study report containing
detailed methodology is freely available at: doi.
org/10.3389/ fimmu.2020.572634.16
Multiplex Assay Kits: This study compared two
multi-analyte detection platforms in the 96well microtiter plate format. The MILLIPLEX MAP
Human High Sensitivity T Cell Magnetic Bead Panel
(#HSTCMAG28SMPX21; Merck EMD Millipore) is a
bead-based fluorescence immunoassay that simultaneously evaluates 21 cytokines.14 The MSD electrochemiluminescence kits were limited to evaluating 10 cytokines maximum each, so we used three
separate kits to achieve reasonable overlap with
the 21-plex LMX assay: MSD Proinflammatory Panel
1 (#K15049G), Cytokine Panel 1 (#K15050G), and
Chemokine Panel 1 (#K15047G).15

Experimental Overview: All assays were performed
by one technologist with more than a decade of
multiplex immunoassay experience.14,15 Thawed
samples were filtered through 0.45 µm syringe filters
and maintained at 4°C. Samples were run in duplicate, and each assay plate contained recombinant
standards for each analyte and kit-provided QC samples. Calibration curves were established using serial 4-fold dilutions of stock QCs. Blank signals were
determined using the sample diluent supplied with
each kit. Samples were diluted 1:4 and processed.

The LMX and MSD assays measured 16 common cytokine analytes: GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6,
IL 7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17, MIP-1α, MIP-1β,
and TNF-α. All assay steps were performed according to manufacturer instructions. To allow maximum
binding time of the analytes for both assays, a 17-hour
primary antibody incubation step was performed according to the kit manuals.14,15 We used the MSD approved overnight incubation instead of the standard
2-hour incubation, due to potential improved assay
sensitivity as indicated by kit instructions.
Detection and Quantitation Systems: Cytokine
level evaluation in LMX plates used a FLEXMAP®
3D instrument operated with xPONENT® Software
v4.2 (both from LMX); MSD plates were read on a
SECTOR® Imager 6000 operated with MSD Discovery
workbench software v3.0/4.0 (both from MSD).

Assay Performance Characteristics: The quantitative accuracy of a multiplex assay depends on the
cumulative quality of individual analyte calibration
curves, which is in turn determined by reagent quality,
appropriate curve-fitting procedures, assay precision,
analyte percentage recovery, and assay LoQs.4,19,20 We
assessed assay precision, lower limits of detection
(LoD), lower and upper limits of quantification (LLoQ
and ULoQ, respectively), and resulting dynamic ranges for cytokine measurement for both assays. The
mean blank values of 28 (LMX) and 22 (MSD) were
used to calculate LoDs, defined as mean blank signal
+3 standard deviations (SD). The LLoQ was either the
analyte LoD or the lowest measurable standard value,
whichever was higher. The ULoQ was defined as the
highest standard curve point in each kit.

Samples: 62 EDTA-anticoagulated human plasma samples were assessed by both assays. Donors

Comparative Quantification of Cytokines in
Human Plasma: Cytokine levels in diverse human

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