Clinical Chemistry 51:7
1102–1109 (2005)
Multiplexed Analysis of Biomarkers Related to
Obesity and the Metabolic Syndrome in Human
Plasma, Using the Luminex-100 System
Mine Y. Liu,1 Antonios M. Xydakis,2 Ron C. Hoogeveen,1 Peter H. Jones,1
E. O’Brian Smith,3 Kathleen W. Nelson,4 and Christie M. Ballantyne1*
Background: The complex pathology of disease has
sparked the development of novel protein expression
profiling techniques that require validation in clinical
settings. This study focuses on multiplexed analyses of
adipocytokines and biomarkers linked to the metabolic
syndrome, diabetes, and cardiovascular disease.
Methods: Multiplexed immunoassays using fluorescent
microspheres and the Luminex-100 system were per-
formed on plasma from 80 obese patients (40 with the
metabolic syndrome) before and after 6 – 8 weeks of
diet-induced weight loss. Leptin, insulin, C-peptide,
monocyte chemoattractant protein-1 (MCP-1), eotaxin,
interleukin-8 (IL-8), tumor necrosis factor-␣ (TNF-␣),
and IL-6 concentrations measured with multiplex panels
from 3 different manufacturers were compared with
results from commercial ELISAs. Detection limits and
between- and within-run imprecision were determined
for each analyte. Bland–Altman analysis was used to
determine agreement between multiplexed immunoas-
says and ELISAs.
Results: Correlation between the Luminex multiplexed
assays and ELISAs was good for leptin (Linco), insulin
(Linco), MCP-1 (Biosource and Upstate), and eotaxin
(Biosource) with correlation coefficients of 0.711– 0.895;
fair for eotaxin (Upstate) and C-peptide (Linco) with
correlation coefficients of 0.496 – 0.582; and poor for
1
Section of Atherosclerosis, Department of Medicine, 2 Division of Endo-
crinology, Diabetes and Metabolism, and 3 Section of Nutrition, Department of
Pediatrics, Baylor College of Medicine, Houston, TX.
4
Methodist Wellness Services, The Methodist Hospital, Houston, TX.
*Address correspondence to this author at: Baylor College of Medicine,
6565 Fannin, M.S. A-601, Houston, TX 77030. Fax 713-798-3057; e-mail
cmb@bcm.tmc.edu.
Received December 17, 2004; accepted April 6, 2005.
Previously published online at DOI: 10.1373/clinchem.2004.047084
1102
Molecular Diagnostics
and Genetics
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TNF-␣, IL-8, and IL-6 (Linco, Biosource, Upstate, and
R&D) with correlation coefficients of ⴚ0.107 to 0.318.
Within- and between-run imprecision values for the
multiplex method were generally <15%. Relative
changes in plasma leptin and insulin concentrations
after diet-induced weight loss were similar whether
assessed by multiplex assay or ELISA.
Conclusion: Although this technology appears useful in
clinical research studies, low assay sensitivity and poor
correlations with conventional ELISA methods for some
analytes with very low plasma concentrations should be
considered when using the Luminex platform in clinical
studies.
© 2005 American Association for Clinical Chemistry
Obesity has reached epidemic proportions in the United
States; ⬃30% of US adults are obese, and the percentage is
increasing (1 ). Obesity increases the risk for the metabolic
syndrome, diabetes, hypertension, atherosclerosis, and
thrombosis (2– 4 ). The National Cholesterol Education
Program Adult Treatment Panel III (ATP III)5 defined the
metabolic syndrome using clinical criteria for abdominal
obesity, triglyceride and HDL-cholesterol concentrations,
blood pressure, and fasting glucose (5 ), and ⬎20% of
adults in the United States meet these clinical criteria (6 ).
Novel technologies such as gene expression arrays have
identified a large number of molecules that have altered
expression in adipose tissue and may be relevant to the
development of the metabolic syndrome, diabetes, and
cardiovascular disease with obesity. In contrast to the
ability to simultaneously examine the concentrations of
thousands of mRNA transcripts, plasma concentrations of
5
Nonstandard abbreviations: ATP III, Adult Treatment Panel III; MCP-1,
monocyte chemoattractant protein-1; TNF-␣, tumor necrosis factor-␣; IL,
interleukin; and NIBSC, National Institute for Biological Standards and Con-
trol.
 Clinical Chemistry 51, No. 7, 2005
cytokines, chemokines, or other biomarkers are usually
measured one at a time by ELISA. Measurement of
plasma concentrations of each putative biomarker with
individual ELISAs incurs considerable time, cost, and
sample volume, which thus limits the ability to systemat-
ically examine the effects of a clinical intervention.
Multiplexed proteomics research may offer a major
advance for clinical research. Protein microarrays are the
most commonly used for simultaneous determination of
multiple proteins in a biological fluid. The technique uses
primary antibodies as the immobilized probe on a solid
surface, and protein antigens labeled with fluorophores
with or without bound secondary antibodies are recog-
nized and detected. However, binding of antibodies and
antigens to a solid support can cause denaturation or
drying of proteins. The Luminex-100 bead-based system
is a recently developed platform that provides multiplex-
ing in a solution phase and thus is particularly flexible
and nondestructive for protein analysis. Each set of up to
100 uniquely color-coded polystyrene microspheres is
anchored with a different capture antibody. The use of
detection antibodies labeled with the fluorochrome R-
phycoerythrin allows quantification of antigen–antibody
reactions that occur on the microsphere surface by mea-
surement of the relative fluorescence intensity. The sys-
tem is capable of measuring potentially up to 100 analytes
simultaneously in a small sample volume (25–50 ␮L). In
theory, the Luminex platform may also provide a wider
dynamic range than conventional ELISA methods be-
cause of the greater linear range of fluorescence intensity
compared with absorbance. The increased dynamic
movement during antibody–antigen reactions that occurs
in solution also may increase its assay efficiency (7–11 ).
The purpose of this study was to compare, in a clinical
research study, several commercially available Luminex
multiplex panels with conventional commercial ELISAs
for measurement, in human plasma, of biomarkers asso-
ciated with obesity and inflammation.
Materials and Methods
participants
From September 2001 to September 2002, a total of 80
obese individuals [56 women and 24 men; mean (SD) age,
47.1 (0.9) years; mean (SD) body mass index, 38.3 (0.7)
kg/m2] were enrolled in a protein-sparing, very low
calorie diet-induced weight loss program. Written in-
formed consent was obtained from each participant. The
weight loss program was approved by the Baylor College
of Medicine Institutional Review Board (12 ). All partici-
pants were obese (body mass index ⬎30 kg/m2), and
one-half had the metabolic syndrome based on the ATP III
criteria (5 ). Analytes were measured in plasma at baseline
for all 80 participants and after 4 – 6 weeks of rapid weight
loss for the 40 individuals with metabolic syndrome
(mean weight loss, 7%). During weight loss, participants
1103
were not started on any medication that would influence
glucose tolerance, insulin secretion, or insulin sensitivity.
collection and storage of blood samples
Blood samples were collected by venipuncture from the
participants into EDTA-containing Vacutainer Tubes after
an overnight fast. The freshly drawn blood was centri-
fuged at 3000g for 20 min at 4 °C. Plasma was then
separated and subsequently stored at ⫺70 °C in small
aliquots. All analyses were performed on frozen plasma
samples.
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luminex multiplex assays
Multianalyte profiling was performed on the Luminex-
100 system and the XY Platform (Luminex Corporation).
Calibration microspheres for classification and reporter
readings as well as sheath fluid were also purchased from
Luminex Corporation. Acquired fluorescence data were
analyzed by the MasterPlexTM QT software (Ver. 1.2;
MiraiBio, Inc.). Plasma concentrations of leptin, insulin,
and C-peptide were determined by the Linco Human
Endocrine 3-Plex Panel (Linco Research, Inc.). Chemo-
kines [monocyte chemoattractant protein-1 (MCP-1) and
eotaxin] were measured by both the Biosource Chemo-
kine 5-Plex Panel (Biosource International, Inc.) and the
Upstate Beadlyte Human Cytokine 8-Plex Panel (Upstate
USA, Inc.). Cytokines [tumor necrosis factor-␣ (TNF-␣),
interleukin-8 (IL-8), and IL-6] were analyzed by the Linco
Human Cytokine 6-Plex Panel (Linco), the Upstate Bead-
lyte Human Cytokine 8-Plex Panel (Upstate), and the
R&D Fluorokine MAP Human Cytokine 6-Plex Panel
(R&D Systems, Inc.). All analyses were performed accord-
ing to the manufacturers’ protocols.
ELISAs
All corresponding ELISAs were performed with commer-
cially available reagent sets. Plasma leptin and C-peptide
were analyzed by use of the Human Leptin ELISA Kit and
the Human C-Peptide ELISA Kit (Linco). Insulin was
assayed by the 1-2-3 Ultrasensitive Human Insulin ELISA
(Alpco Diagnostics, American Laboratory Products Com-
pany). MCP-1 and eotaxin were measured by the Quan-
tikine Human Eotaxin Immunoassay and Quantikine Hu-
man MCP-1 Immunoassay (R&D Systems). TNF-␣ and
IL-6 were measured by the Quantikine HS Human TNF-␣
Immunoassay and the Quantikine HS Human IL-6 Immu-
noassay (R&D Systems). The circulating IL-8 concentra-
tion was measured by the Biosource Human IL-8 Immu-
noassay (Biosource International).
detection limits and recovery study
To determine the detection limits for the Luminex assays,
3 SD were added to the mean fluorescence intensity of 2
zero calibrator replicates. The respective analyte concen-
trations were then calculated from the calibration curve.
Similarly, to evaluate the detection limits for the ELISAs,
3 SD were added to the mean absorbance value of 2 zero
1104 Liu et al.: Luminex Multiplexed Analysis of Human Plasma

calibrator replicates, and the respective analyte concentra-
tions were calculated from the calibration curves. The
recombinant protein MCP-1 and IL-6 calibrators provided
with the ELISA and Luminex reagents were added into
normal healthy human plasma. The plasma was manu-
facturer pooled with specific concentrations of lipids
requested (lot no. BC0208A; Pacific Biometrics, Inc.).
statistical analysis
Statistical analyses were performed with SPSS data anal-
ysis software (Ver. 11.5; SPSS Inc.). Analyze-it software
(Ver. 1.71; Analyze-it Software Ltd.) was used for Deming
panel was not yet calibrated against the WHO standard
and no WHO standard for eotaxin is currently available, it
was not possible to compare the detection limits of the
Biosource panel with ELISAs. Unlike the Biosource panel,
the Upstate multiplex panel has a significantly higher
detection limit for MCP-1 than the ELISA from R&D.
Cytokines TNF-␣, IL-8, and IL-6 in 3 different multi-
plex panels from different manufacturers (Linco, Upstate,
and R&D) were compared with ELISAs. In general, the
ELISAs tended to have lower detection limits.
percentage of samples with undetectable

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regression and Passing–Bablok analyses for method com-
parisons to account for imprecision in both the ELISA and
Luminex methods. The Wilcoxon signed-rank test was
used to analyze the effect of rapid weight loss on leptin
and insulin. Bland–Altman analyses (13 ) were performed
to calculate limits of agreement and systematic errors, and
the results were plotted with y as the value of the
difference (⌬) between Luminex and ELISA, x as the mean
of the Luminex and ELISA values, and the upper and
lower limits defined by the mean ⌬ ⫾ 2 SD.
Results
detection limits of luminex assays and ELISAs
A comparison of detection limits for the Luminex assays
and the ELISAs is shown in Table 1. Some analyte
concentrations were also converted to the National Insti-
tute for Biological Standards and Control (NIBSC)/WHO
international reference standard unit (IU/L) if calibra-
tions of the assays were performed by the manufacturers.
In the Linco Endocrine multiplex panel, leptin and C-
peptide had significantly lower detection limits by the
Luminex assay. The Alpco ultrasensitive insulin ELISA
was more sensitive for detection of insulin than the Linco
Endocrine multiplex.
Two chemokine panels (Biosource and Upstate) were
chosen to compare with ELISAs for the analysis of MCP-1
and eotaxin. Because the MCP-1 assay in the Biosource
MCP-1, TNF-␣, IL-6, and IL-8 concentrations in
the luminex assays and ELISAs
We compared the percentage of samples in which 4
representative analytes could not be detected by the
Luminex and ELISA methods; the results are shown in
Table 2. For the ELISAs, generally the percentage of
samples with undetectable analyte concentrations was
0%, except for IL-8 in the Biosource assay, which is not a
high-sensitivity assay. For the Luminex assay, in the
Upstate multiplex panel, nearly 50% of samples had
undetectable concentrations of IL-6 and TNF-␣; in the
R&D multiplex panel, nearly 100% of samples had unde-
tectable IL-6, TNF-␣, and IL-8; and in the Biosource
multiplex panel, 61% of samples had undetectable IL-8.
assay precision of luminex and ELISA methods
The between- and within-run imprecision, as the CV
(CV ⫽ SD/mean), for the Luminex assays is shown in
Table 1 of the Data Supplement (available at http://
www.clinchem.org/content/vol51/issue7/) and in Table
3. The Upstate multiplex panel and 3 plasma pools from
patients with metabolic syndrome with low, medium, and
high concentrations of added analyte were chosen to test
the between-run precision. The 2 normal control plasma
samples (QA and QC) were not selected because they
were undetectable by the multiplex panel. The MCP-1
between-run imprecision, determined from 7 multiplex

Table 1. Comparison of detection limits for the Luminex multiplexed assays and the individual ELISAs.
Multiplex panels Analytes Luminex ELISA ELISA manufacturer
Linco Endocrine Leptin 0.13 ␮g/L 1.39 ␮g/L Linco
Insulin 0.31 mIU/L 0.14 mIU/L Alpco
C-peptide 0.037 ␮g/L 0.132 ␮g/L Linco
Biosource Chemokine MCP-1 2.35 ng/L 2.57 ng/L R&D
Eotaxin 4.37 ng/L 3.80 ng/L R&D
Upstate Chemokine MCP-1 20.5 ng/L (0.023 kIU/L) 2.57 ng/L (0.002 kIU/L) R&D
Eotaxin 27.72 ng/L 3.80 ng/L R&D
Linco Cytokine TNF-␣ 1.61 ng/L (0.249 kIU/L) 0.092 ng/L (0.0028 kIU/L) R&D
IL-8 2.52 ng/L (0.026 kIU/L) 0.26 ng/L (0.0018 kIU/L) Biosource
Upstate Cytokine TNF-␣ 0.065 ng/L (0.0019 kIU/L) 0.092 ng/L (0.0028 kIU/L) R&D
IL-8 0.70 ng/L (0.0003 kIU/L) 0.26 ng/L (0.0018 kIU/L) Biosource
IL-6 0.75 ng/L (0.1029 kIU/L) 0.068 ng/L (0.0084 kIU/L) R&D
R&D Cytokine TNF-␣ 1.53 ng/L (0.052 kIU/L) 0.092 ng/L (0.0028 kIU/L) R&D
IL-6 0.977 ng/L (0.1280 kIU/L) 0.068 ng/L (0.00843 kIU/L) R&D
 Clinical Chemistry 51, No. 7, 2005 1105

Table 2. Samples with undetectable concentrations of MCP-1, TNF-␣, IL-6, and IL-8 in the ELISAs and the Luminex
multiplexed assays.
IL-6 TNF-␣ MCP-1 IL-8

Assay manufacturer % undetectable n % undetectable n % undetectable n % undetectable n

ELISAs
R&D 0 40 0 40 0 120 NAa NA
Biosource NA NA NA NA 40.5 40
Luminex system
Upstate 47.4 154 51.7 114 10.3 154 0 120
R&D 100 40 95.2 40 NA NA 100 40
Linco 16.7 40 7.1 40 NA NA 21.4 40

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Biosource NA NA NA NA 2.4 40 61 40
a
NA, not applicable.

assays with each sample analyzed in duplicate, was validation of the luminex method in human
⬎15%. The within-run precision represents the mean plasma
values of 3 samples with low, medium, and high concen- We chose ELISAs for comparison with the newer Lumi-
trations of the analyte that were analyzed 20 times in 1 nex technique because the ELISA is a generally accepted
assay. The within-run CVs for all analytes ranged from method for analyzing biomarkers in human serum or
6.6% to 12% and were within the acceptable limit. plasma. Two methods of comparison between Luminex
The between- and within-run precision of the ELISAs assays and ELISAs were carried out. We first used linear
are shown in Tables 1 and 3 of the online Data Supple- correlation to evaluate the relationship between Luminex
ment. The between-run CV was determined from 2 assays and ELISAs. We then applied the Bland–Altman
plasma control samples (QA and QC) because they were method (13 ) to assess the agreement between the 2
detectable by the high-sensitivity ELISAs. For each sam- measurement methods.
ple, the CV was determined from the means of 6, 8, and 5
assays for MCP-1, TNF-␣, and IL-6, respectively, and were Correlation. The results of the Deming regression analysis
analyzed in duplicate. The within-run precision was cal- comparing the Luminex and ELISA methods are shown in
culated from the mean CVs of n samples analyzed in Table 4. The correlation coefficient indicates scatter of the
duplicate. Overall, the within-run CVs were within rea- measured data about the Deming regression line, and it is
sonable limits, ranging from 5.0% to 15%, except for IL-6 determined by the precision of the 2 assay methods. Table
(21%). For the high-sensitivity ELISAs, the between-run 4 shows the results for the Linco endocrine panel. The
imprecision generally indicated a ⬎15% CV of random correlation coefficients (R) were 0.812, 0.895, and 0.582 for
errors, and the within-run imprecision showed a ⬎15% leptin, insulin, and C-peptide, respectively. The correla-
CV of random errors. tion coefficient for C-peptide was 0.67 after omission of
the 2 outliers (defined as values outside 3 SD). In the
Deming regression analysis, the slope indicates a propor-
tional error that might have originated from incomplete
Table 3. Within-run assay imprecision. recovery in one of the methods or from different accura-
Analytes Within-run CV, % n
cies of the calibrators used. The slopes were 1.28, 2.38, and
Multiplexed panels
0.68 for leptin, insulin, and C-peptide, respectively. The
Linco Endocrine Leptin 11 120 smallest proportional error was for leptin, i.e., the values
Insulin 6.6 120 obtained from the 2 assay methods were the least differ-
C-Peptide 10 80 ent. The intercept indicates a systematic error that might
Biosource Chemokine MCP-1 12 40 be caused by interference, such as insufficient blank
Eotaxin 10 40 compensation. The intercepts were 2.26 ␮g/L, ⫺3.03
Upstate Chemokine MCP-1 8.1 40 kIU/L, and ⫺1.83 ␮g/L for leptin, insulin, and C-peptide,
Eotaxin 11 40 respectively. The smallest systematic error was for C-
ELISAs peptide.
Linco Leptin 5.9 120 The correlation coefficients were 0.263 and 0.899 for
Alpco Insulin 15 40 MCP-1 in the Biosource panel with and without the 2
Linco C-Peptide 10 40 outliers, respectively; outliers were defined as values
R&D MCP-1 5.4 120
outside 3 SD. For eotaxin, the correlation coefficient was
R&D Eotaxin 5.0 120
0.711 for the Biosource panel. The slopes for MCP-1
1106 Liu et al.: Luminex Multiplexed Analysis of Human Plasma

Table 4. Deming regression results for comparison of the multiplexed assays performed on the Luminex systems (y) and
individual ELISAs (x).
Slope Intercept
a b
Analyte Deming Sy円x R Coefficient SE 95% CI Mean SE 95% CI
Leptin (␮g/L) 9.90 0.812 1.28 1.28 1.11–1.44 2.26 3.19 ⫺4.05 to 8.57
Insulin (mIU/L) 5.62 0.895 2.38 0.19 1.99–2.77 ⫺3.03 3.47 ⫺10.06 to 4.00
C-Peptide (␮g/L) 2.32 0.582 0.68 0.15 0.37–0.99 ⫺1.83 1.20 ⫺4.25 to 0.59
Eotaxin (ng/L) 20.44 0.711 7.71 1.29 5.10–10.32 ⫺49.85 80.64 ⫺213.55 to 113.86
MCP-1c (ng/L)
With outliers 110.14 0.263 40.44 25.09 ⫺10.49 to 91.36 ⫺7829.64 6421.28 ⫺20 865.54 to 5206.25
Without outliers 49.99 0.899 4.97 0.42 4.11–5.82 79.60 109.36 ⫺142.90 to 302.09

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a
The manufacturers of the assays for the Luminex multiplex systems (y) and the individual ELISAs (x) were as follows: for leptin, Linco for both; for insulin, Linco
and Alpco, respectively; for C-peptide, Linco for both; for eotaxin, Biosource and R&D, respectively; and for MCP-1, Biosource and R&D, respectively.
b
CI, confidence interval.
c
Outliers are defined as values outside 3 SD of the mean for 2 replicates of the zero calibrators.

without the outliers and for eotaxin were 4.97 and 7.71 for
the Biosource panel, respectively, and the intercepts were
79.60 and ⫺49.85 ng/L, respectively. Passing–Bablok
analysis revealed both proportional and constant bias for
MCP-1 and eotaxin (data not shown).
We also analyzed the correlations between the Lumi-
nex assays and ELISAs for the cytokines TNF-␣, IL-8, and
IL-6. The correlation coefficients were 0.104 and ⫺0.107
for TNF-␣ and 0.266 and 0.318 for IL-8 in the Linco and
Upstate panels, respectively. The correlation coefficient
was 0.298 for IL-6 in the Upstate panel. Overall, the correla-
tions between Luminex assays and ELISAs for the 3 cyto-
kines analyzed were poor in all the multiplex panels.
Bland–Altman analysis. The difference between the Lumi-
nex assay and the ELISA was plotted against the mean of
the Luminex assay and ELISA for each plasma sample
measured. The Bland–Altman plots for several analytes
are shown in Fig. 1. The Bland–Altman analysis was
carried out only for (a) analytes that showed good corre-
lation and used the same calibrators for calibration curves
in both the Luminex assays and ELISAs (leptin, insulin,
and C-peptide) or (b) assays already calibrated against the
WHO standards (MCP-1 and insulin).
The Bland–Altman plots for leptin, insulin, and C-
peptide in the Linco endocrine panel and MCP-1 in the
Upstate panel are shown in Fig. 1. The systematic differ-
ences between the 2 assay methods as calculated by the
Bland–Altman analysis were 11.0 ␮g/L, 13.8 kIU/L, 3.9
␮g/L, and 0.21 kIU/L for leptin, insulin, C-peptide, and
MCP-1, respectively. The 2 methods agreed fairly well for
leptin values ⬍50 ␮g/L. Above 50 ␮g/L, however, the
Luminex assays gave both higher and lower values than
the ELISA; the discrepancy for some measurements was
as large as 75%. For leptin, 7.5% of the patients had values
that differed by ⬎2 SD. Agreement between 2 methods is
generally acceptable if ⬍5% of values differ by ⬎2 SD. For
insulin, the 2 assays agreed very well at concentrations
⬍30 mIU/L, but above 30 mIU/L, the Luminex assay
gave higher values than the ELISA, although only 2.5% of
the values differed by ⬎2 SD. For C-peptide, most of the
Luminex measurements gave lower values than the
ELISA (mean difference, 3.9 ␮g/L). At concentrations ⬍4
␮g/L, the 2 methods agreed very well, and only 2.5% of
the values differed by ⬎2 SD. For MCP-1 in the Biosource
panel, the 2 methods agreed well at concentrations ⬍0.5
kIU/L, and 5.5% of patients had values that differed by
⬎2 SD. For insulin, C-peptide, and MCP-1, the only
measurements that differed by ⬎2 SD were the extreme
highest values of the analytes for patients in this study.
recovery study
Recovery experiments detect inaccuracy of a method
resulting from systematic errors. The results of the recov-
ery study for MCP-1 in both the ELISA and Luminex
assay are shown in Table 2 of the online Data Supplement.
The recombinant protein calibrators provided with the
ELISA and the Luminex assay were added at 3 different
concentrations in pooled normal healthy human serum
for measurement of recovery with 10 replicates or more.
The 3 concentrations (32, 128, and 512 ng/L) of MCP-1
were selected because they were commonly seen in our
study in healthy individuals and in patients with meta-
bolic syndrome. The pools containing added Luminex
calibrator were measured by the Luminex method, and
the pools containing the added ELISA calibrator were
measured by ELISA. As shown in Table 2 of the online
Data Supplement, for the pools containing 128 and 512
ng/L MCP-1, the ELISA gave mean recoveries of 98.8%
and 102.1%, respectively. However, in the Luminex assay,
the mean recoveries of MCP-1 were 58.2%, 49.1%, and
54.6%, respectively, for the 3 concentrations.
effects of active weight loss on leptin and
insulin in individuals with the metabolic
syndrome
The patients with metabolic syndrome lost 7% of their
initial weight [8.0 (0.55) kg, or 17.6 (1.2) pounds] after 4 – 6
 Clinical Chemistry 51, No. 7, 2005 1107

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Fig. 1. Bland–Altman analysis showing the agreement between Luminex assays and ELISAs for the measurement of leptin (A), insulin (B), C-peptide
(C), and MCP-1 (D).
The y axis in each panel represents the difference (⌬) between Luminex and ELISA values, and the x axis represents the average of the Luminex and ELISA values.
Solid lines are mean values; dotted lines are 2 SD. For leptin (A), the mean difference is 11.0 ␮g/L (2 SD ⫽ 29.7 ␮g/L); for insulin (B), the mean difference is 13.8
mIU/L (2 SD ⫽ 37.4 mIU/L); for C-peptide (C), the mean difference is ⫺3.9 ␮g/L (2 SD ⫽ 6.8 ␮g/L); and for MCP-1 (D), the mean difference is 0.21 kIU/L (2 SD ⫽
0.99 kIU/L).

weeks of the diet-induced weight loss program. Plasma
concentrations of leptin and insulin at baseline and after
weight loss were measured by both the Luminex and
ELISA methods, and the results were compared. Mean
(SD) leptin concentrations measured by ELISA were sig-
nificantly higher before weight loss compared with after
weight loss [36.3 (21.5) vs 19.8 (18.7) ␮g/L; n ⫽ 40; P
⬍0.001, Wilcoxon signed-rank test]. The Luminex assay
also showed a significant mean (SD) difference after
weight loss [47.4 (25.6) vs 27.6 (22.1) ␮g/L; n ⫽ 40; P
⬍0.001]. Insulin concentrations were also significantly
higher before weight loss than after weight loss whether
measured by ELISA [19.2 (17.3) vs 9.4 (7.8) mIU/L; n ⫽
13; P ⫽ 0.039] or the Luminex assay [37.4 (41.1) vs
14.7 (16.0) mIU/L; n ⫽ 13; P ⫽ 0.028]. The percentage
changes in leptin and insulin after weight loss are shown
in Fig. 1 of the online Data Supplement. The median
percentage changes in leptin were ⫺54% and ⫺46% as
measured by the ELISA and Luminex assay, respectively,
and the median percentage changes in insulin were ⫺41%
and ⫺56%, respectively.
comparison of MCP-1 between healthy
controls and metabolic syndrome patients
with 4 or more risk factors
We compared plasma MCP-1 concentrations in healthy
controls (n ⫽ 14) and metabolic syndrome patients with 4
or more risk factors (n ⫽ 20). The MCP-1 concentrations
measured by ELISA and the Luminex assay are shown in
Fig. 2 of the online Data Supplement. The mean (SD)
MCP-1 concentrations measured by ELISA were signifi-
cantly higher in the metabolic syndrome patients than in
healthy controls [536.5 (316.1) vs 263.3 (96.5) ng/L; P
⬍0.001, Wilcoxon signed-rank test]. The median MCP-1
values measured by ELISA were 414.6 and 231.4 ng/L for
patients and controls, respectively. The results obtained
with the Luminex assay also indicated that the mean (SD)
MCP-1 concentrations in the metabolic syndrome patients
1108 Liu et al.: Luminex Multiplexed Analysis of Human Plasma
were significantly higher than those in healthy controls
[83.5 (101.1) vs 20.4 (8.6) ng/L; P ⬍0.001, Wilcoxon
signed-rank test]. In the Luminex assay, the median
MCP-1 values were 63.9 and 18.4 ng/L for patients and
controls, respectively.
Discussion
Multiplex immunoassays using a small sample volume
could facilitate clinical research in complex disorders such
as the metabolic syndrome and could be extremely help-
ful in the evaluation of changes in biomarkers with
therapeutic interventions. The detection limits shown in
Table 1 allow for a potentially high degree of detectability
in the patients’ samples. However, because the focus of
this study was to compare the validity of Luminex-based
assays vs traditional ELISAs as a tool for clinical research,
the use of functional sensitivity is a more meaningful
index than limit of detection, and in our clinical study,
many cytokines were not detectable in a high percentage
of patient plasma samples by the Luminex multiplex
method. The inability to detect these analytes by the
multiplex method may be explained in part by interfer-
ences, such as heterophilic antibodies, that are present in
complex sample matrices such as plasma.
The within-run imprecision (CV) values obtained for
the multiplex method were generally ⬍15%, whereas the
between-run imprecision for the multiplex method for
MCP-1 was ⬎15%. The large interassay variations seen
with MCP-1 should be considered when using multiplex
panels for this analyte (and potentially others) in clinical
research, for example, by measuring all samples in 1 run
(or at least all the samples from a specific individual
before and after an intervention), and by including control
sample pools in all runs.
The poor correlations between the Luminex assay and
ELISAs for the 3 cytokines analyzed in our study are most
likely attributable to the extremely low plasma concentra-
tions of these cytokines and the presence in blood of
interfering substances such as heterophilic antibodies,
which are known to interfere with immunoassays (14, 15 ).
Furthermore, our results show an increase in variability at
lower concentrations for both methods (Luminex and
ELISA), which also may partially explain the poor corre-
lations between the 2 methods.
Our studies demonstrate that in our laboratory the
multiplexed assays for leptin (Linco), insulin (Linco),
MCP-1 (Biosource and Upstate), and eotaxin (Biosource)
showed good correlations of ⬎0.7 (correlation coefficients
ranging from 0.711 to 0.895); eotaxin (Upstate) and C-
peptide (Linco) showed fair correlations of 0.5– 0.7 (corre-
lation coefficients ranging from 0.496 to 0.582); and
TNF-␣, IL-8, and IL-6 (Linco, Biosource, Upstate, and
R&D) showed very poor correlations (⬍0.5) with ELISAs
(correlation coefficients ranging from ⫺0.107 to 0.318).
The changes in plasma concentrations of leptin and insu-
lin after diet-induced weight loss showed similar percent-
age reductions as assessed by multiplex assays or ELISA
(Fig. 1 of the online Data Supplement). The relative
difference in plasma concentrations of MCP-1 between
controls and metabolic syndrome patients also were sim-
ilar in the 2 methods (Fig. 2 of the online Data Supple-
ment). The total error acceptable for an assay depends on
the clinical setting in which the assay is being used. For
example, in clinical research, studies are frequently de-
signed to examine the association of plasma concentra-
tions of analytes with the disease phenotype (in this case,
obese patients with the metabolic syndrome vs lean
controls) and the impact of a therapeutic intervention on
the disease state (such as weight loss). In the present
Downloaded from https://academic.oup.com/clinchem/article/51/7/1102/5630020 by guest on 27 February 2021
study, measurements of MCP-1 by ELISA or Luminex
assay provided similar information about the relative
increase in plasma concentrations associated with obesity
and the metabolic syndrome compared with lean controls,
and the relative changes in leptin and insulin concentra-
tions before and after weight loss were similar by either
ELISA or Luminex assay. However, the “total error” of an
assay that is acceptable to assess relative change in a
clinical research setting may be far greater than what is
acceptable in a clinical practice setting, which is focused
on diagnosis and treatment of disease.
Our study has important differences in design com-
pared with previous reports that have validated multi-
plexed immunoassays, which measured IgG antibodies
(16 ), lipopolysaccharide-stimulated human plasma sam-
ples (8 ), or supernatant from stimulated cells (17 ). A
recent study reported the use of Luminex-based multiplex
analysis to measure 9 cytokines in sera from 30 children
infected by rotavirus and 9 healthy control children, but
the investigators did not validate their findings against
conventional ELISAs (18 ). A different study used dried
blood, spotted on paper, from children with cerebral
palsy and showed rather poor correlation between the
Linco and Upstate multiplex panels (19 ).
In conclusion, although this technology appears useful for
analytes present in a wide range of concentrations in
plasma, low detectability and poor correlations with con-
ventional ELISA methods for some analytes when present
in very low concentrations in plasma should be consid-
ered when using the multiplexed assay platform in clini-
cal studies. Furthermore, improvement in the detection
limits for some of the analytes that have very low plasma
concentrations and are currently commercially available
for Luminex-based multiplex assays is crucial for their use
in analysis of unstimulated blood samples. Issues such as
the effects of interfering heterophilic antibodies, efficiency
of capture antibody-coupling techniques, development of
high-sensitivity multiplex assays, and optimization of
capture/detection antibody pairing need to be studied
further to improve the efficacy of this technology for use
in clinical studies. Manufacturers should calibrate their
secondary standards to the primary NIBSC/WHO stan-
dards, consistent with the recommendations for immuno-
assay standardization described in the report of Wadhwa
 Clinical Chemistry 51, No. 7, 2005
and Thorpe (20 ). However, our data, as well as results
from a growing number of research studies, demonstrate
that multiplex technology may be useful in clinical re-
search to measure a large number of analytes to examine
the association with a clinical phenotype and the effects of
therapeutic interventions, and that this technology may
be particularly useful when sample volume is limited,
such as in large epidemiologic studies and clinical trials.
This study was supported by Applied Technology Grant
No. 004949-0093-2001 from the state of Texas and an
American Diabetes Association Research Award. The
lipid laboratory is supported by donations from George
and Cynthia Mitchell, Nijad Fares, and Jeffrey Hines. The
Upstate Beadlyte Human Cytokine 8-Plex Panel was
kindly provided by Upstate USA, Inc. (Lake Placid, NY),
The IL-8 ELISA was a gift from Biosource International,
Inc. (Camarillo, CA). We also appreciate the technical
assistance and useful discussions of Dr. Laurie Stephen of
Upstate USA, Inc., and the helpful comments of Henry
Pownall, PhD, of the Section of Atherosclerosis, Baylor
College of Medicine.
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