1. Methods
The comparison data for three of the six lupus nephritis panel analytes
(MCP-1, NGAL, and adiponectin) is shown below. Samples were run both on
ELISA and Luminex simplex assays to determine how well the ELISA data
correlated with Luminex data prior to assembling the analytes into a panel.
Pearson correlation coefficients were 0.88 (p=0.04) for NGAL, 0.89 (p<0.00001)
for MCP-1 and 1.0 (p<0.00001) for adiponectin.
• The levels of the renal biomarkers are measured by Luminex Magpix assays
and compared to ELISA data for 16 randomly chosen samples.
• The multiplex assay we generated was compared to commercial ELISA kits
for human NGAL (BioPorto Diagnostics, Denmark), human MCP-1, and
human adiponectin (the latter two from R&D Systems, Minneapolis, MN).
• Initially all kits were used according to their individual manufacturers
recommendations. NGAL and MCP-1 bead sets continue to follow the
manufacturers protocol; however, due to dilution constraints, we used a
different dilution factor as our standard for adiponectin, keeping the other two
assays regular standard curve intact.
• Data from these were compared to the data generated from the multiplex on
the same day. To assemble the three bead sets into one assay, we used the
ProcartaPlex Human Basic Kit (eBioscience, Vienna, Austria) for the liquid
medium.
• ELISAs were compared to these singleplex magnetic bead assays, prior to
combining the assays into a triplex.
• Data was analyzed using Spearman rank order.
Potential Biomarkers for Predicting Lupus Nephritis
Activity in Children
OBJECTIVE. The purpose of this study is to compare the results
of multiplex assays to published results of enzyme-linked
immunosorbent assays (ELISAs) for a panel of six biomarkers,
hemopexin, ceruloplasmin, neutrophil gelatinase-associated lipocalin
(NGAL), kidney injury molecule-1 (KIM-1), monocyte chemoattractant
protein-1 (MCP-1), and adiponectin. Previous studies have identified
these biomarkers to have the potential to predict lupus nephritis activity.
This study aims to develop multiplex assays for established panels of
these SLE nephritis biomarkers.
METHODS. The levels of
the renal biomarkers are
measured by multiplex
assays and compared to published ELISA data.
This was initially done by
comparing magnetic bead assays with ELISA data
from the same day.
Magnetic bead assays were combined with minimal changes to
manufacturers recommendations for further comparison with the
corresponding ELISAs.
RESULTS. The data for three of the six analytes shows that there
was not a significant difference between ELISA data and the data
collected from the multiplex assays for NGAL, MCP-1, and adiponectin
(Pearson correlation coefficients were 0.88 (p=0.04) for NGAL, 0.89
(p<0.00001) for MCP-1 and 1.0 (p<0.00001) for adiponectin.)
CONCLUSION. This allows us to begin creating an established
multiplex panel, to which we can compare future samples. This will make
the process of determining biomarker levels faster and more cost
effective, so that we can better predict lupus activity. As we continue with
this study, we will test the remaining three analytes. Although a
promising start, further research with a larger sample size is required to
validate the Luminex multiplex technology.
Results
Discussion
References
Introduction
Caroline B. Christian, Christopher Haffner, Qing Ma, Kasha Wiley, Michael R. Bennett, Hermine I. Brunner
Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine
Singleplex vs. ELISA
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Mishra, and Prasad Devarajan. "Urinary Neutrophil Gelatinase–associated Lipocalin as a Biomarker of
Nephritis in Childhood-onset Systemic Lupus Erythematosus." Arthritis & Rheumatism Arthritis Rheum 54.8
(2006): 2577-584. Web.
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Gitelman, Judyann Olson, Karen Onel, Kathleen M. O'neil, Earl D. Silverman, Lori Tucker, Jun Ying, Prasad
Devarajan, and Hermine I. Brunner. "Initial Validation of a Novel Protein Biomarker Panel for Active Pediatric
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Pendl, David Witte, Jun Ying, Brad H. Rovin, and Prasad Devarajan. "Association of Noninvasively Measured
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• Systemic lupus erythematosus (SLE) affects approximately 1.5 million
Americans. Many of these individuals develop kidney disease (lupus
nephritis).
• There is a known connection between lupus activity and renal function
in patients with a lupus nephritis diagnosis.
• The current biomarkers used to predict lupus activity and damage are
expensive, invasive, and insensitive.
• Previous research has identified six promising renal biomarkers,
hemopexin, ceruloplasmin, NGAL, KIM-1, MCP-1, and adiponectin,
which are being assembled into a multiplex assay.
• Previous work has been done using ELISAs; however, the major
disadvantages of the ELISAs are cost, time, and required sample
volumes.
• Multiplex assays minimize these factors by saving antibody, sample,
and cost per analyte, and include a greater dynamic range.
• By comparing the multiplex assays of MCP-1, NGAL, and adiponectin
to ELISA data we are able to begin developing multiplex assays for
established panels of kidney disease markers.
• The expectation is that this will allow doctors to better predict lupus
nephritis activity, and thus treat it more effectively.
• Our data indicates that there was not a significant difference
between the data from multiplex assays and ELISA methods
described previously in publication
• This information has the potential to simplify the process of testing
samples so that we can better predict changes in lupus nephritis
activity.
• There are limitations of this study to consider when reviewing the
data.
1. Human error may allow for slight variations in singleplex data.
2. Also, data was collected from a relatively small population, in
which random chance is more likely to play a role.
3. The study collected information only from patients who were
under the age of 18, so this information may not be consistent
in adult populations.
• Another key concern with working with
multiplex bead assays is the possibility of
the “matrix effect,” which is why we chose
to use singleplex assays for our initial
comparisons.
• A multiplex composed of these three
analytes has also been conducted; however,
the results have not been compared to
ELISA data from the same day.
• As we continue with this study, we will
examine hemopexin, ceruloplasmin, and
KIM-1, by comparing them to the accepted
ELISA data.
• Future research we plan to conduct will
include translational studies incorporating
this new information after the completed
assay is validated.
• In conclusion, these results are promising
for patients suffering from lupus nephritis,
but further research is required to validate
this technology and rule out chance factors.
Abstract