Quantitative, High-Multiplex and Label-Free Detection of
Serum Cytokines and Inflammatory Markers on the Arrayed
Imaging Reflectometry Platform
Carter JA, Keller PC, Taran JB, Striemer CC, Miller BL
Adarza Research and Development: 150 Lucius Gordon Drive, Ste. 110, West Henrietta, NY 14586
Adarza Business Operations and Manufacturing: 501 Pearl Drive, Ste. K70-1, St. Peters, MO 63376
Abstract
New multiplex, label-free methods for the sensitive and
quantitative detection of serum proteins are highly desirable, as
they have the potential to dramatically accelerate biomedical
research by generating more information at lower cost than
currently available techniques. We have used Arrayed Imaging
Reflectometry (AIR™), a label-free protein microarray
technology that relies on the target binding-induced
perturbation of an antireflective coating on the surface of a
silicon chip, to create a multiplex antibody array for the
detection of human serum cytokines and inflammatory markers.
The chip is able to provide quantitative information about
protein concentrations, with limits of detection in picograms per
mL and high dynamic range. Compatible with standard 96-well
microplate protocols, the label-free nature of the array will
permit expansion to arbitrary-plex size and simple creation of
custom panels as there are no secondary antibodies or labels to
cross-react.
Table 2. Detection performance for the 25-Plex AIR Array
Detection Performance (pg/mL)
TARGET
ULLOD
Automated Microarray Image Processing
Expose
to Target
Captured single image
Segmentation of probe spots
ULLOQ
LLOD
Average Recovery
(%)
Average CV (as %)
5-PL Fit (R^2)
LLOQ
CRP
5000000
5000000
<12.8
<64
94.1
12.43
0.9962
EGF
200000
200000
<12.8
<12.8
104.4
6.39
0.9867
Eotaxin
200000
200000
<12.8
<12.8
103.0
9.79
0.9901
Fractalkine
200000
200000
<12.8
<12.8
96.8
5.66
0.9815
GM-SCF
200000
200000
<12.8
<12.8
84.9
8.43
0.9902
IFN-g
200000
200000
<2.6
<2.6
118.3
9.47
0.9988
IL-11
200000
200000
<12.8
<64
105.1
14.98
0.9964
IL-12p70
200000
200000
<2.6
<12.8
92.7
8.64
0.9638
IL-16
200000
200000
<2.6
<2.6
97.0
9.44
0.9861
IL-1a
200000
200000
<12.8
<12.8
102.0
7.60
0.9981
IL-1b
200000
200000
<12.8
<12.8
80.0
7.66
0.9670
IL-2
200000
200000
<2.6
<12.8
112.9
15.56
0.9965
IL-29
200000
200000
<12.8
<12.8
96.4
11.84
0.9952
IL-6
200000
200000
<12.8
<12.8
120.9
8.78
0.9883
IL-7
200000
200000
<2.6
<2.6
121.4
11.16
0.9821
IL-8
200000
200000
<2.6
<64
112.2
6.78
0.9961
MCP-2
200000
200000
<12.8
<12.8
115.0
10.51
0.9868
MCP-3
200000
200000
<12.8
<64
106.7
9.32
0.9934
MCP-4
200000
200000
<12.8
<12.8
119.3
17.19
0.9909
MDC
200000
200000
<12.8
<12.8
90.8
11.06
0.9521
MIP-1a
200000
200000
<12.8
<320
79.2
6.99
0.9952
RANTES
200000
200000
<12.8
<64
101.0
12.04
0.9887
TARC
200000
200000
<12.8
<64
104.7
18.93
0.9988
TNFα
200000
200000
<2.6
<12.8
112.5
15.74
0.9801
TRAIL
200000
200000
<2.6
<2.6
84.6
12.75
0.9898
(Upper limits of quantitation are 200 ng/mL, save CRP which is 5 µg/mL)
Pixel-level spot analysis
0.652605
2
2
4
4
6
6
8
8
2
4
6
Results
0.660893
8
Calibration
curves
2
0.661002
4
6
8
10
8
10
0.738697
2
2
4
4
6
6
8
8
10
Target
Conc.
10
2
4
6
8
2
4
6
Figure 2. Inter-assay detection performance for three panel constituents.
Conclusions
Figure 1. A representative 25-plex assay standard curve series. Note;
for all 8-point standard curves a 5-parameter logistic fit is used.
www.adarzabio.com
Adarza has developed a pre-production, novel, label-free multiplex
immunoassay with comparable performance in human serum to current
commercially available ELISA and bead-based technologies. Current product
development is focused on further improving the precision, limits of detection,
and overall analytic performance characteristics of the assay, as well as
expanding the plex size to span every known human cytokine and chemokine.
Adarza’s AIR™ Technology finally delivers on the promise of label free:
• Multiplexing up to 400 Plex in one well of 96 well plate.
• Superior Precision and Accuracy
• Limits of Detection and Quantitation to 1 pg/mL
• Seven (7) Logs of Dynamic Range across entire assay panel
• Walk – away instrument operation
• Significant cost per data-point and time to result advantages