Development of a single assay to detect multiple melanoma markers using an electrochemiluminescence platform. Researchers successfully developed individual assays for CEACAM, GDF-15, and MIF. They then optimized a multiplex assay on a Meso Scale Discovery platform to detect these 4 proteins (CEACAM, GDF-15, MIF, and IL-8) in a single well with low cross-reactivity. Testing demonstrated the assay could accurately detect all proteins using a small sample volume, making it a promising tool for monitoring treatment response and survival prediction in melanoma patients.

1. Development of a single assay to detect multiple melanoma
markers using an electrochemiluminescence platform
Ryan Lynch 1, Stephan Ariyan 2, AntonellaBacchiocchi 3, Harriet Kluger 4, Mario Sznol 4,
and Ruth Halaban 3
1Maine
Institute for Human Genetics & Health
2Department of Surgery, Yale University School of Medicine
3Department of Dermatology, Yale University School of Medicine
4Comprehensive Cancer Section of Medical Oncology,
Yale University School of Medicine
Abstract Developing Individual Assays
Recent advances in protein detection instrumentation have allowed for the accurate
Of the four proteins identified as potential candidates for a multiplex assay, CEACAM,
quantitation of minimally-expressed proteins in complex matrices, such as human serum.
GDF-15, and MIF had not been developed into an MSD format. MIHGH developed these
Meso Scale Discovery (MSD) has developed a protein detection platform that utilizes a
three MSD-based assays using an antibody-sandwich assay (figure 3). These assays
combination of electrochemilumenescence and multiplexing capabilities to produce results
utilized biotinylated primary detection antibodies incubated with MSD Sulfo-Tag®
which are low in background and highly sensitive over a broad dynamic range. Another
Streptavidin secondary detection antibodies. Conditions that were optimized included
advantage offered on the MSD platform is the ability to multiplex, in which multiple proteins
concentrations of capture and detection antibodies, sequential addition of primary and
can be detected in a single well of a 96 or 384 well-plate format. One such multiplexing
secondary detection antibodies, diluents, blocking reagents, and plate binding capacities.
application was developed at the Maine Institute for Human Genetics and Health (MIHGH)
With conditions optimized using recombinant proteins, separate experiments were run to
in a project sponsored by the Yale Specialized Program of Research Excellence (YSPORE)
determine whether the analytes were detected in human plasma samples, and also to gain an
in Skin Cancer. Researchers at the YSPORE recently identified a panel of eight proteins as
understanding of the normal ranges and dilution factors required. Lower limits of detection
potential markers for tumor load in patients with melanoma: sICAM, OPN, TIMP-1, MIA,
(LLOD) of low pg/mL were obtained with a dynamic range of >3-logs concentration.
CEACAM, GDF-15, IL-8, and MIF. The panel could be used to track patient response to
Calculated concentration CVs for plasma samples were generally <5%.
treatment and to predict survival. Of the eight proteins, four have been identified by MIHGH
as candidates for multiplexing on the MSD platform: CEACAM, GDF-15, IL-8 and MIF.
MIHGH successfully developed and optimized 4 unique electrochemiluminescent assays
utilizing a 96-well plate format for the individual proteins of interest. An antibody-sandwich CEACAM MIF GDF-15
approach was employed in which a bare well was coated with: 1. Unconjugated antibody; 2. pg/mL pg/mL pg/mL
Calc. Low 14.4 Calc. Low 2.82 Calc. Low 0.63
Recombinant protein; 3. Biotinylated detection antibody bound to a streptavidin detection Calc. High
Calc. Low
10000
27.8
Calc. High
Calc. Low
8000
7.52
Calc. High
Calc. Low
10000
3.78
Calc High 8000
antibody, which is conjugated with MSD’s SulfoTag antibody (by which ruthenium emits Calc High 10000 Calc High 10000
light when electrically charged). Optimization methods included screening multiple
antibody pairs for each protein, using different antibody and protein/sample diluents,
detection of recombinant and native protein, and evaluating different antibody
concentrations. Using these optimized conditions, capture antibodies corresponding to the
four proteins were printed onto each well of a 96-well plate. Extensive testing and
optimization by MIHGH demonstrates the new assay’s capability to detect the panel of four
proteins in a single well with a very low level of antibody cross-reactivity. This assay is
capable of detecting very low levels of all four proteins with a small amount of sample Figure 3. Standard-bind plate (blue) and high-bind plate (red). Plasma samples from healthy human donors are shown
required for accurate results (typically less than 50 μL per sample). at multiple dilutions.
Multi-Array® Technology Developing a Single Multiplex
MIHGH utilized Multi-Array® technology developed by Meso Scale Discovery
(Gaithersburg, MD) in designing these assays. MSD’s Multi-Array® technology is a Assay to Detect 4 Analytes
proprietary combination of electrochemiluminescence detection and patterned arrays. Results obtained in these experiments indicate that CEACAM, GDF-15, MIF and IL-8 (an
Electrochemiluminescence detection uses labels that emit light when electrochemically assay developed by MSD) would work well as a single multiplexed assay. These assays all
stimulated. Background signals are minimal because the stimulation mechanism (electricity) require similar sample dilutions and diluents. Prototype standard-bind plates with four spots
is decoupled from the signal (light). Multiple excitation cycles of each label amplify the per well were printed by MSD using a proprietary process. Each capture antibody was
signal to enhance light levels and improve sensitivity. coated at 100 μg/mL. MIHGH tested several conditions of the plates: cross reactivity and
non-specific interactions of capture and detection antibodies capture antibody specificity;
and the specificity of the detection analyte (Tables 1 & 2). The plates were tested using
purified recombinant proteins.
Blocker A Average Signal Average Bkg % CrossReactivity
Calibrator IL8 CEACAM MIF GDF 0 0 0 0 IL8 CEACAM MIF GDF
Detection IL8 CEACAM MIF GDF IL8 CEACAM MIF GDF IL8 CEACAM MIF GDF
Spot
CEAMCAM 387.50 22912.50 232.50 190.00 140.00 6351.50 171.50 94.00 0.40 100.00 0.33 0.44
GDF 274.50 219.00 172.00 22086.00 56.00 161.00 83.50 58.00 0.35 0.35 0.47 100.00
MIF 279.50 165.00 18777.00 165.50 49.00 102.50 102.50 51.00 0.37 0.38 100.00 0.52
IL8 61729.50 209.00 147.50 160.50 143.00 140.50 69.00 47.50 100.00 0.41 0.42 0.51
Dil 2/3 Average Signal Average Bkg % CrossReactivity
Calibrator IL8 CEACAM MIF GDF 0 0 0 0 IL8 CEACAM MIF GDF
Detection IL8 CEACAM MIF GDF IL8 CEACAM MIF GDF IL8 CEACAM MIF GDF
Spot
CEAMCAM 313.00 4737.50 101.50 193.50 83.50 190.00 80.00 101.50 0.20 100.00 0.06 0.25
GDF 274.00 104.00 95.50 37510.00 58.50 86.50 58.50 62.00 0.19 0.38 0.10 100.00
MIF 228.00 83.50 35408.50 202.00 55.50 58.00 134.50 46.50 0.15 0.56 100.00 0.42
IL8 114481.00 99.00 82.50 187.00 70.00 94.00 77.50 72.50 100.00 0.11 0.01 0.31
Table 1. Cross reactivity of calibrator on other spots.
Calibrator IL-8 CEACAM
Fig. 1. Electrochemiluminescence detection. Used with permission from MSD. Detection IL8 CEACAM MIF GDF IL8 CEACAM MIF GDF
Spot
CEAMCAM -0.01 -0.03 -0.01 -0.01 0.27 100.00 0.25 0.18
GDF -0.01 -0.01 -0.01 -0.01 0.13 -0.17 0.09 0.12
MIF 0.03 0.00 -0.03 0.00 -0.07 0.08 -0.41 0.00
IL8 100.00 -0.01 0.00 0.00 0.02 -0.09 -0.01 -0.02
MSD’s Multi-Spot plates offer multiple arrays within the well, enabling the multiplexing of
Calibrator MIF GDF
up to 10 analytes in a single well. Detection IL8 CEACAM MIF GDF IL8 CEACAM MIF GDF
Spot
CEAMCAM -0.07 -0.24 -0.02 -0.09 -0.01 -0.06 -0.01 0.03
GDF 0.00 -0.05 0.01 -0.02 0.00 -0.02 0.00 100.00
MIF 50.87 52.59 100.00 47.52 0.00 -0.01 -0.05 0.08
IL8 -0.02 -0.03 0.00 -0.01 -0.01 -0.02 -0.01 0.12
Table 2. Cross reactivity of detection antibodies.
Overall, the percent of cross reactivity was very low (<0.5%). Testing of the plates is
ongoing.
Fig. 2. 96-well Multi-Spot plate with 4 spots per well.
Acknowledgements
Funding was provided by the Yale SPORE in Skin Cancer. W. VanScyoc, Ph.D, MSD
Copyright 2011 Maine Institute for Human Genetics & Health. Applications Scientist, provided technical assistance.