APPLICATION NOTE

Analyzing Crohn’s Disease Biomarkers Utilizing Bio-Techne’s
Immunoassay Platforms: From Multiplex Discovery onLuminex® to Single Analyte Analysis with Quantikine® ELISAsINTRODUCTION
Crohn’s disease is an inflammator y bowel disease (IBD) thataffects an estimated 1.6 million people in the United Statesalone according to the Crohn’s and Colitis Foundation of America(CCFA). It primarily affects the gastrointestinal system and has
long been recognized as a formidable disease with highrecurrence rates even after surgical treatment is attempted.
Anastomotic recurrence occurs endoscopically as early as
several months after bowel resection, followed by symptomaticrecurrence; reoperation is then often required.
Multiple clinical biomarkers of Crohn’s Disease have been studied
and developed into tests like serum C-Reactive Protein (CRP) andfecal calprotectin (FCP). Although there have been some studies
to correlate biomarkers with endoscopic scores, relapses, andpostoperative recurrence prediction, their main value thus far is in
managing patients with Crohn’s disease. Therefore, identifying novel, accurate circulating biomarkers is aresearch priority.
R&D Systems, a Bio-Techne Brand, offers a variety of specific and reproducible immunoassay platforms thatmake it easy to discover, analyze, and verify novel biomarkers and automate your favorite biomarker research.
In this application note, we take you through a proof-of-concept example of how to streamline your biomarker
studies by using R&D Systems’ Luminex® assays for your biomarker discovery and Quantikine® ELISAs for yourbiomarker validation and subsequent analysis.
Luminex assays are ideal for biomarker discovery when you have samples with limited volume but want tomonitor expression of many targets. It allows for simultaneous quantification of up to 50 analytes in just 3.5hours with as little as 25–50 µL of sample. Luminex multiplex panels give you more data points per sample andare fully customizable so you’ll have total control over the targets screened. Once you have potential
biomarkers identified, you can validate your results and perform long-term studies with Quantikine ELISAs.
Quantikine ELISAs are perfect for both validation and long-term studies when monitoring protein expression
level or response to treatment. They are the most published ELISAs on the market and deliver the mostaccurate and precise results. R&D Systems has over 30 years of expertise developing ELISA assays, ensuring
long term assay consistency and reproducibility even between different assay lots.

1

A

MATERIALS AND METHODS

100000Crohn’s patient samples were purchased from BioIVT andapparently healthy patient samples collected in-house. Samples
were first analyzed with the R&D Systems Luminex assays (HumanXL Cytokine Discovery Panel, Catalog # LKTM014 and HumanLuminex Assay, Catalog # LXSAHM) to screen a large panel oftargets, some known to have different expression levels according
to published literature. A select subset of the identified markers
were then validated using in the same samples with either
Quantikine Colorimetric Sandwich ELISAs or Quantikine HighSensitivity Colorimetric Sandwich ELISAs when appropriate(Catalog numbers below). Assays were all tested as recommended
in the product insert.

Healthy Serum
Crohn’s Serum

Concentration (pg/mL)

10000100010010**CD
40LigandEGFL Eo F
T- tax
3L inFr igaac nta dlkin
GR e
OGR α
OIL β-1raIL-6ILTN 8FαIP-1M 0CP
M -1IP-3
PD MI αGF P-3-A β8/BBVE
GF

1RESULTSB

We first used the Luminex system to screen circulating levels of 48
cytokines in Crohn’s and healthy serum samples. The screen
identified multiple cytokines with either similar or elevated serum
levels in disease samples compared to apparently healthy samples(Figure 1). Levels of IL-6 and TNF-α were below the limit ofquantification (LOQ) in the healthy serum samples, while some of
the Crohn’s serum samples were detectable. Large differenceswere observed in IL-8, CRP, and GRO-α with a 21.8-fold, 5.4-fold,
and 3.5-fold increase in the Crohn’s samples, respectively.

10 8

Concentration (pg/mL)

Selected cytokines from the Luminex testing which exhibited
increased levels in Crohn’s disease samples were further analyzed
on Quantikine ELISA kits. The same serum samples were tested induplicate on either Quantikine or High Sensitivity Quantikine
ELISAs (Table 1) for analytes with low circulating levels (Figure 2).
All analytes were detected within LOQ for both healthy anddiseased serum samples. Average sample coefficient of variance(CV) was below 4% for all samples in all assays. Quantikine results
were consistent with Luminex data, demonstrating the ability toeasily move from one platform to the other as you progress
through the biomarker workflow and your long-term research.

Target

Healthy Serum
Crohn’s Serum

10 710 610 510 410 310 2
10 1

CRPICAM-1

IL-18

Figure 1. Selected targets from a Luminex assays comparing cytokine
levels in serum samples from healthy and Crohn’s disease patients.
Target concentrations were interpolated using a standard curve andaveraged. Serum samples were diluted 1:2 and tested on the
Human XL Cytokine Discovery Panel (Catalog # LKTM014) (A).
Serum samples were also diluted 1:2 (ICAM-1, IL-18) or 1:200(CRP) and tested on a Human Luminex Assay (Catalog # LXSAHM)(B). Graph includes the target mean and standard error of the mean
(SEM). (*) = target below LOQ.

Kit

Catalog #Standard Curve Range
0.781–50 ng/mL

CRP

Quantikine Colorimetric Sandwich ELISA

DCRP00

ICAM-1

Quantikine Colorimetric Sandwich ELISA

DCIM000.625–40 ng/mL

IL-1raQuantikine® Colorimetric Sandwich ELISA

DRA00

31.3–2000 pg/mL

IL-18

Quantikine® Colorimetric Sandwich ELISA

DL180

15.6–1000 pg/mL

IL-6

Quantikine HS® Colorimetric Sandwich ELISAs

HS600C

0.156–10 pg/mL

IL-8

Quantikine HS Colorimetric Sandwich ELISAs

HS800

1.0–64 pg/mL

TNF-α

Quantikine HS Colorimetric Sandwich ELISAs

HSTA00E

0.156–10 pg/mL®
®

®
®

Table 1. Crohn’s disease and healthy patient samples run on Quantikine kits to validate Luminex assay
data.
2CONCLUSIONS

10 8

Healthy Serum
Crohn’s Serum

10 7

Concentration (pg/mL)

Discovering novel biomarkers for a variety of conditions including
Crohn’s Disease, cancer, and neurodegenerative disease is crucial
to develop new treatments, monitor disease progression, andmeasure treatment response. Bio-Techne offers a diverse portfolio
of immunoassay platforms and assays to optimize your biomarker
research workflow which give you fast, reproducible data whileconserving your precious samples.
To demonstrate this workflow, we screened over 48 cytokines inhealthy vs. Crohn’s disease serum samples using the Luminex
platform. The data was ready in approximately 3.5 hours. From
this data we identified 7 targets to evaluate further with BioTechne’s legendary Quantikine ELISA product line. The data
between the Luminex platform and the Quantikine ELISA’s
correlated well, demonstrating the ability to move between assay
techniques depending on experimental criteria. Whether you are atan initial screening stage or biomarker validation, Bio-Techne has
the solution to provide you with accurate and reproducible results.

10 610 510 4
10 3
10 2
10 1
10 0
10 -1

CRPICAM-1

IL-1ra

IL-18

IL-6

IL-8

TNF-α

Figure 2. Sample quantification using Quantikine Colorimetric andQuantikine High Sensitivity Colorimetric assays. Graph includes thetarget mean and standard error of the mean (SEM).

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