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Incorporation of SOMAmer® Reagents in Single Molecule Array (Simoa) Assays for Cytokine Detection with Ultra-high Sensitivity Danlu Wu, Evaldas Katilius, Edgar Olivas, Milena Dumont Milutinovic, and David R. Walt Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02451 • Publication Date (Web): 16 Aug 2016 Downloaded from http://pubs.acs.org on August 19, 2016
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Incorporation of SOMAmer® Reagents in Single Molecule Array (Simoa) Assays for Cytokine Detection with Ultra-high Sensitivity Danlu Wu,† Evaldas Katilius,‡ Edgar Olivas,‡ Milena Dumont Milutinovic,† David R. Walt *†
†
Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford MA 02155 USA
‡
SomaLogic, Inc, 2945 Wilderness Place, Boulder, CO 80301, USA *Corresponding author: Phone/fax: 617-627-3470 /617-627-3443. Email:
[email protected] ABSTRACT: Slow Off-rate Modified Aptamers (SOMAmers) are attractive protein recognition reagents due to their high binding affinities, stable chemical structures, easy production and established selection process. Here, biotinylated SOMAmer reagents were incorporated into single molecule array (Simoa)-based assays in place of traditional detection antibodies for six cytokine targets. Optimization and validation were conducted for TNF-α as a demonstration using a capture antibody/detection-SOMAmer detection scheme to highlight the performance of this approach. The optimized assay has a broad dynamic range (>four log10 units) and an ultralow detection limit of 0.67 fM (0.012 pg/mL). These results show comparable sensitivity to our antibody pair-based Simoa assays, and tens to thousands-fold enhancement in sensitivity compared with conventional ELISAs. High recovery percentages were observed in a spike-recovery test using human sera, demonstrating the feasibility of this novel Simoa assay in detecting TNFα in clinically-relevant samples. Detection SOMAmers were also used to detect other cytokines, such as IFN-γ, IL-1β, IL-2, IL-6 and IL-10, in human samples. Although not yet demonstrated, in principle it should be possible to eventually replace both the capture and detector antibodies with corresponding SOMAmer pairs in sandwich immunoassays. The combination of the ultrasensitive Simoa platform with the higher reliability of SOMAmer binding reagents will greatly benefit both biomarker discovery and disease diagnostic fields.
binding reagents.4-6 In contrast to conventional DNA or RNA aptamers, SOMAmer reagents contain modified nucleotides.7-10 With functional moieties (e.g., benzyl, 2-napthyl, or 3-indolylcarboxamide) conjugated at the uracil ring that mimic amino acid side chains, SOMAmer reagents resemble antibodies in their ability to bind target proteins.6,11,12 SOMAmer reagents are selected through a novel version of the Systematic Evolution of Ligands by EXponential enrichment (SELEX) protocol that involves evaluating candidates from large libraries containing modified nucleotides.8,13 After rigorous purification and characterization, they have stable chemical structures with high nuclease resistance and binding affinities that are comparable to antibodies.13 Importantly, SOMAmer reagents are produced by solid-state DNA synthesis methods. Therefore the purity and yields can be reproducibly controlled. Customized SOMAmer reagents for a desired protein target can be produced relatively rapidly and economically compared to antibodies. As chemically stable as DNA, SOMAmer reagents in buffer solution are able to tolerate multiple freezethaw and heating-cooling cycles with minimal degradation.6 They have already been utilized in various research applications, such as Clostridium difficile toxins detection,4 blood-based cancer diagnosis,5 and biomarker discovery for chronic kidney disease.13 SOMAmer sandwich pair selection and sandwich assays have been also demonstrated.14 As a result, SOMAmer reagents possess great potential for replacing antibodies in immunoassays for protein detection.
In enzyme-linked immunosorbent assays (ELISAs), the target recognition reagents, typically antibodies, play the key role in the assay performance of most assays. In a typical sandwich-type ELISA, the capture antibody (Cap-Ab), which is usually attached to a solid surface, specifically captures the antigen, followed by addition of a second antibody (detection antibody, Det-Ab) that binds the antigen at a different epitope than the Cap-Ab. Thus the antigen is “sandwiched” between the two antibodies. Immunoassay specificity is thus enhanced by two independent binding events and the assay performance is highly dependent on the binding affinity of antibodies.1 Normally, the production of antibodies is accomplished by firstly injecting target antigen samples into laboratory or farm animals. High expression levels of antigenspecific antibodies are then produced in the animal serum. Polyclonal antibodies are recovered directly from serum, while monoclonal antibodies are produced through a much stricter and more tedious process involving hybridoma cell isolation and antibody purification.2,3 Although the technology for producing antibodies has been extensively developed and improved in recent decades, the use of antibodies in detection application is still limited by their potential for cross-reactivity, difficult production process, high cost and poor reproducibility.1 Consequently, it is highly desirable to seek new molecular recognition reagents with comparable specificity and binding affinity to antibodies, but also with more reproducible and simpler production processes. A new class of versatile ligands, SOMAmer (Slow Off-rate Modified Aptamer) reagents, have emerged as promising protein-
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Figure 1. (A) Schematic illustration for Cap-Ab/Det-SOMAmer based Simoa assay. (B) Simoa standard curve for TNF-α. A fourparameter logistical curve (1/y2 weighted) was applied for fitting with ε > 0.99. (C) Cross-reactivity test for TNF-α with 11 cytokines ([cytokine] = 1 pM). Assays in B and C were performed in SB17T with 25%NBCS. [Det-SOMAmer] = 50 nM, [Z-block] = 50 µM, [SβG] = 100 pM. (D) Simoa assay results for six cytokines. Lines are plotted to connect points for guiding the reader. Assays are performed in SB17T with 0.5%BSA. [Det-SOMAmer] = 10 nM, [SβG] = 150 pM. All the assays in B, C and D were performed as a 2-step format with 1st incubation time = 15 min and 2nd incubation time = 5 min. Error bars are shown for triplicate measurements. spike-recovery tests in real human samples. The high recovery percentages obtained demonstrate the great potential of this assay for clinically-relevant sample detection with ultra-high sensitivity. We believe, after similar systematic optimizations, that substitution of detection SOMAmers for detection antibodies in Simoa assays is promising for other cytokines, such as IL-2, IL-1β, IFNγ, IL-6 and IL-10. Our previously reported antibody-based Simoa assay for cytokine detection involved a 3-step incubation protocol.18 However, we found that a 2-step incubation protocol is more favorable for the Cap-Ab/Det-SOMAmer-based assay, as suggested by a TNF-α assay performed with various conditions and protocols, shown in Figure S-1. Both the human recombinant cytokines and DetSOMAmer reagents were prepared in the assay buffer SB17T with 0.5% BSA as the blocking reagent (Experimental Section in Supporting Information). For the 3-step assay, based on the reported 3-step antibody pair-based Simoa assays,22 we sequentially incubated the Cap-Ab immobilized beads with target molecules (15 min), Det-SOMAmer reagents (5, 10, or 15 min) and enzyme (5 min) with three washes between each incubation step. The 2step assay requires the beads, target proteins and Det-SOMAmer reagents to be incubated simultaneously (15 min), followed by the enzyme labeling incubation (5 min) (Figure 1A). Under the same assay conditions (buffer formulation, reagent concentration), a much higher signal to noise ratio (S/N) was obtained for the 2step assay, although the background was slightly higher (Figure S-1). Presumably, the SOMAmer reagent is able to recognize and bind a freely diffusing target protein more easily than a Cap-Abprotein complex in which some epitopes on the protein are already occupied. Consequently, further optimization and validation of the novel SOMAmer-based Simoa assay were conducted in the 2-step format. Further optimization was carried out using TNF-α as a demonstration by evaluating the signal responses from [TNF-α] = 50 fM and blank analyte samples to assess specific and nonspecific binding, respectively. The experimental parameters were tuned by varying the parameters of one assay component while keeping other components fixed. The duration of the 1st incubation was optimized using the following conditions: [Det-SOMAmer] = 10 nM, 2nd incubation time = 5 min, and [SβG] = 150 pM. The best S/N value at [TNF-α] = 50 fM was observed when the 1st incubation time = 15 min (Figure S-2). Next, by fixing the 1st incubation time at 15 min and maintaining other assay conditions, the second incubation time was optimized. A 5-min 2nd step incubation yielded the highest S/N and relatively low background (Figure S-3). When selecting the optimal SβG concentration, both 100 pM and 150 pM exhibited very high responses at [TNF-α] = 50 fM, but we found
Cytokines, known for their role as intercellular mediators, perform a critical function in shaping and maintaining the immune response.15 The importance of sensitively measuring cytokines at low concentration, especially in the early stages of disease, has attracted increased attention.16,17 Our group has previously reported detection of cytokines in the aM-fM range by employing antibody pairs as recognition reagents on a single molecule array (Simoa) platform, which combines microwell arrays with ELISA technology.18 Similar to traditional ELISA kits, the antibody pairbased sandwich immunocomplexes are formed on magnetic microbeads, followed by labeling with streptavidin-conjugated enzyme (Streptavidin-β-galactosidase, SβG). In contrast to conventional ELISAs, in the Simoa assay, each bead is loaded, isolated and sealed into individual microwells with extremely small volumes (~ 40 fL) on a microwell array platform in the presence of enzyme substrate.19 Fluorescent products generated by the enzymatic reaction are confined within the microwells, ensuring high local fluorescence intensity that can be clearly recorded by a CCD. By employing digital analysis for samples in the low protein concentration range and analog analysis for samples in the high concentration range, the Simoa assays, with signal response expressed as average enzyme per bead (AEB), can detect proteins down to the sub-fM concentration level while maintaining a wide dynamic range of up to four logs.20 The whole assay process can be conducted in an automatic fashion with the HD-1 Analyzer, which was produced based on Simoa technology by the Quanterix Corporation.21 In our experience with developing and applying Simoa assays, we found extensive variation among commercial antibodies from different lot numbers, even when they are derived from the same gene code and, to the best of our knowledge, produced via the same technology. The inconsistency between antibody batches has occasionally affected the reliability of our Simoa assays,18 motivating us to seek alternative binding reagents with greater reliability and reproducibility. In the current study, we demonstrate the feasibility of incorporating SOMAmer reagents as alternative binding reagents in the Simoa platform. In our approach, the Cap-Ab is immobilized on the para-magnetic microbeads to capture the target cytokine, while the biotinylated SOMAmer reagent (Det-SOMAmer) substitutes for a biotinylated Det-Ab as the secondary recognition reagent. Therefore, a novel antibody-protein-SOMAmer complex is formed on the bead. After careful assay optimization, this new capture antibody/detection-SOMAmer (Cap-Ab/Det-SOMAmer)based Simoa assay is developed for detecting TNF-α with a detection limit (LOD) and dynamic range comparable to those of antibody-based Simoa assays, and much better than those of conventional ELISA kits. As part of assay validation, we have performed
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that 100 pM was preferred due to the lower background noise (Figure S-4). In order to apply this assay to detect TNF-α in serum samples, newborn calf serum (NBCS), which mimics a human serum matrix, was added into SB17T to constitute the standard buffer. The recombinant human cytokine was spiked into SB17T with 25% NBCS. To minimize non-specific background signal, polyanionic competitor (also called Z-block) was added to the buffer. We explored the optimal Z-block conditions to minimize non-specific interactions without compromising the sensitivity of the assay. The highest S/N and optimal background levels were observed when [Det-SOMAmer] = 50 nM and [Z-block] = 50 µM (Table S2 and S-3). Utilizing the optimal assay conditions, a calibration curve was thus created by plotting AEB response against calibrator concentration (0–6 pM) as shown in Figure 1B. A four parameter logistic (4PL) model was applied for fitting (fitting coefficient = 0.999). The calculated LOD for TNF-α is 0.67 fM (3SD method), which is comparable to that of the antibody-based Simoa assay (~0.72 fM) 18 and much lower than those of conventional antibody pair-based ELISAs, which are usually in pM or pg/mL range. The LOQ was roughly calculated as 0.85 fM through precision profile by reading the calibrator back off the calibration curves obtained in three individual runs (Figure S-5). Consequently, the dynamic range of this Cap-Ab/Det-SOMAmer Simoa assay spans over at least four logs (0.0034–24 pM), which fulfills the requirement for clinical sample detection without needing further optimization. 18 The specificity of the Cap-Ab/Det-SOMAmer-based Simoa assay was characterized by cross-reactivity tests with ten commonly measured serum cytokines: GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, and IL-10. Recombinant human proteins of TNF-α and ten other cytokines were spiked at 1 pM in SB17T with 25% NBCS. A blank sample and the 11 cytokine samples underwent a 2-step Simoa assay using the established optimal conditions in the HD-1 Analyzer. As depicted in Figure 1C, all off-target cytokines exhibited signals equivalent to the background signal, while the TNF-α signal is 180-fold higher than the background, suggesting excellent selectivity for TNF-α from other cytokines. The strong performance of this TNF-α assay is promising for further development of SOMAmer-based multiplexed assays, which demand low cross-reactivity and high specificity. To validate the application of our optimized assay in detecting TNF-α in real serum samples, a spike and recovery test was performed. A group of samples was prepared by spiking a series of TNF-α concentrations into human sera collected from different individuals. The samples were diluted with SB17T buffer and then measured in triplicate and concentrations of TNF-α in nonspiked and spiked samples were back-calculated using the calibration curve in Figure 1B. The % recovery data were computed using the following formula: − % = 100 ×
source of variation, the NBCS buffer used in our calibration measurements may not fully mimic human serum. It is likely that by conducting an additional dilution on the samples, this matrix effect can be reduced. Further investigation on the calibration medium should be conducted before applying these assays to clinical samples. Table 1. Results for spike-recovery test Sample No.
1
2
3
4
5
6
Here, is the detected concentration of target molecule in the sample, is the background value (original concentration of target molecule in the sample before spiking) and is the spiked concentration in the samples. The % recovery values for each concentration and relative standard deviations (RSDs) are listed in Table 1. The average % recovery is 104.2%, and most of the values are within a reasonable range of accuracy (70% to 130%), suggesting the developed assay is promising for sample detection. During the test, we found that the % recovery varied among human sera from different individuals. As one possible
CSpike (fM)
CDet (fM)
CDet-C0 (fM)
Recovery (%)
RSD (n = 3)
0.0
1125.6
0.0
-
3.2
6.4
1131.6
6.1
95.0
13.1
12.5
1134.6
9.0
72.0
2.6
25.0
1161.6
36.0
144.0
4.9
50.0
1180.6
55.0
110.0
13.8
100.0
1240.6
115.0
115.0
4.0
200.0
1333.2
207.6
103.8
13.9
400.0
1525.7
400.1
100.0
2.5
800.0
1690.3
564.8
70.6
5.1
0.0
573.6
0.0
-
13.9
14.8
585.8
12.2
82.4
9.5
44.4
609.3
35.7
80.4
4.0
133.2
768.8
195.1
146.5
4.9
400.0
1045.1
471.5
117.9
2.6
0.0
1586.6
0.0
-
2.1
44.4
1642.7
56.1
126.4
3.6
133.2
1752.5
165.9
124.6
7.2
400.0
1965.8
379.2
94.8
1.3
0.0
777.6
0.0
-
1.7
200.0
1024.0
246.4
123.2
3.6
400.0
1160.9
383.3
95.8
1.7
800.0
1585.2
807.6
100.9
4.5
0.0
876.5
0.0
-
6.7
200.0
1054.1
177.6
88.8
6.4
400.0
1245.1
368.6
92.1
3.5
800.0
1759.5
883.0
110.4
0.2
0.0
1858.3
0.0
-
5.7
100.0
1967.3
109.0
109.0
3.4
400.0
2272.2
413.9
103.5
2.6
800.0
2601.6
743.2
92.9
1.3
To demonstrate that the Cap-Ab/Det-SOMAmer Simoa assay format is also applicable to other cytokines, Simoa assays were demonstrated for five additional cytokines in assay buffer SB17T with 0.5% BSA as the blocking reagent. Figure 1D shows the Simoa assay results for TNF-α, IFN-γ, IL-1β, IL-2, IL-6 and IL10 with the best matched Cap-Ab and Det-SOMAmer for each cytokine (Table S-1). All five of these cytokine assays exhibited excellent performance comparable to the TNF-α assay, with
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REFERENCES (1) Zichi, D.; Eaton, B.; Singer, B.; Gold, L. Curr. Opin. Chem. Biol. 2008, 12, 78-85. (2) Steiner, L. Nature 1989, 341, 32-32. (3) Liu, N.; Zhao, Z. Y.; Tan, Y. L.; Lu, L.; Wang, L.; Liao, Y. C.; Beloglazova, N.; De Saeger, S.; Zheng, X. D.; Wu, A. B. Anal. Chem. 2016, 88, 1246-1252. (4) Ochsner, U. A.; Katilius, E.; Janjic, N. Diagn. Microbiol. Infect. Dis. 2013, 76, 278-285. (5) Min, M. R.; Chowdhury, S.; Qi, Y.; Stewart, A.; Ostroff, R. Pac. Symp. Biocomput. 2014, 87-98. (6) Kraemer, S.; Vaught, J. D.; Bock, C.; Gold, L.; Katilius, E.; Keeney, T. R.; Kim, N.; Saccomano, N. A.; Wilcox, S. K.; Zichi, D.; Sanders, G. M. PLoS One 2011, 6, e26332. (7) Tuerk, C.; Gold, L. Science 1990, 249, 505-510. (8) Vaught, J. D.; Bock, C.; Carter, J.; Fitzwater, T.; Otis, M.; Schneider, D.; Rolando, J.; Waugh, S.; Wilcox, S. K.; Eaton, B. E. J. Am. Chem. Soc. 2010, 132, 4141-4151. (9) Brody, E. N.; Willis, M. C.; Smith, J. D.; Jayasena, S.; Zichi, D.; Gold, L. Mol. Diagn. 1999, 4, 381-388. (10) Rohloff, J. C.; Gelinas, A. D.; Jarvis, T. C.; Ochsner, U. A.; Schneider, D. J.; Gold, L.; Janjic, N. Mol. Ther. Nucleic Acids 2014, 3, e201. (11) Tuerk, C.; Gauss, P.; Thermes, C.; Groebe, D. R.; Gayle, M.; Guild, N.; Stormo, G.; Daubentoncarafa, Y.; Uhlenbeck, O. C.; Tinoco, I.; Brody, E. N.; Gold, L. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 1364-1368. (12) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818-822. (13) Gold, L.; Ayers, D.; Bertino, J.; Bock, C.; Bock, A.; Brody, E. N.; Carter, J.; Dalby, A. B.; Eaton, B. E.; Fitzwater, T.; Flather, D.; Forbes, A.; Foreman, T.; Fowler, C.; Gawande, B.; Goss, M.; Gunn, M.; Gupta, S.; Halladay, D.; Heil, J.; Heilig, J.; Hicke, B.; Husar, G.; Janjic, N.; Jarvis, T.; Jennings, S.; Katilius, E.; Keeney, T. R.; Kim, N.; Koch, T. H.; Kraemer, S.; Kroiss, L.; Le, N.; Levine, D.; Lindsey, W.; Lollo, B.; Mayfield, W.; Mehan, M.; Mehler, R.; Nelson, S. K.; Nelson, M.; Nieuwlandt, D.; Nikrad, M.; Ochsner, U.; Ostroff, R. M.; Otis, M.; Parker, T.; Pietrasiewicz, S.; Resnicow, D. I.; Rohloff, J.; Sanders, G.; Sattin, S.; Schneider, D.; Singer, B.; Stanton, M.; Sterkel, A.; Stewart, A.; Stratford, S.; Vaught, J. D.; Vrkljan, M.; Walker, J. J.; Watrobka, M.; Waugh, S.; Weiss, A.; Wilcox, S. K.; Wolfson, A.; Wolk, S. K.; Zhang, C.; Zichi, D. PLoS One 2010, 5, e15004. (14) Ochsner, U. A.; Green, L. S.; Gold, L.; Janjic, N. Biotechniques 2014, 56, 125-128. (15) O'Gorman, M. R. G.; Donnenberg, A. D. Handbook of human immunology, 2nd ed.; CRC Press: Boca Raton, 2008; p 623. (16) Gorelik, E.; Landsittel, D. P.; Marrangoni, A. M.; Modugno, F.; Velikokhatnaya, L.; Winans, M. T.; Bigbee, W. L.; Herberman, R. B.; Lokshin, A. E. Cancer Epidemiol. Biomarkers Prev. 2005, 14, 981-987. (17) Burska, A.; Boissinot, M.; Ponchel, F. Mediators Inflamm. 2014, ID 545493. (18) Wu, D. L.; Milutinovic, M. D.; Walt, D. R. Analyst 2015, 140, 6277-6282. (19) Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S. C.; Fournier, D. R.; Song, L.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J.; Ferrell, E. P.; Randall, J. D.; Provuncher, G. K.; Walt, D. R.; Duffy, D. C. Nat. Biotech. 2010, 28, 595-599. (20) Rissin, D. M.; Fournier, D. R.; Piech, T.; Kan, C. W.; Campbell, T. G.; Song, L.; Chang, L.; Rivnak, A. J.; Patel, P. P.; Provuncher, G. K.; Ferrell, E. P.; Howes, S. C.; Pink, B. A.; Minnehan, K. A.; Wilson, D. H.; Duffy, D. C. Anal. Chem. 2011, 83, 2279-2285. (21) Wilson, D. H.; Rissin, D. M.; Kan, C. W.; Fournier, D. R.; Piech, T.; Campbell, T. G.; Meyer, R. E.; Fishburn, M. W.; Cabrera, C.; Patel, P. P.; Frew, E.; Chen, Y.; Chang, L.; Ferrell, E. P.; von Einem, V.; McGuigan, W.; Reinhardt, M.; Sayer, H.; Vielsack, C.; Duffy, D. C. J. lab Autom. 2016, 21, 533-547. (22) Gaylord, S. T.; Dinh, T. L.; Goldman, E. R.; Anderson, G. P.; Ngan, K. C.; Walt, D. R. Anal. Chem. 2015, 87, 6570-6577.
measured LODs = 0.5 fM for IL-2, IL-6 and IL-1β, and LODs = 1 fM for IL-10 and IFN-γ (Table S-1). The results suggest that, after systematic optimization and development, these assays should also be useful for human sample analysis. In summary, Simoa assays were developed using biotin-labeled SOMAmer reagents in place of Det-Abs. SOMAmer reagents are preferable to antibodies due to their high reliability, structural stability, reproducibility, easy production and established selection processes. The optimization and characterization of the CapAb/Det-SOMAmer-based Simoa assay for TNF-α was successfully demonstrated. We have shown that SOMAmer reagents can be used as detection reagents to create novel cytokine detection assays with ultra-high sensitivity, broad dynamic range, and excellent performance. In particular, its excellent performance was evidenced by the high % recovery in spike-recovery tests in human serum samples. We believe, after similar assay optimization, that SOMAmer reagents will be able to replace the Det-Abs in Simoa assays for other cytokines, such as IFN-γ, IL-1β, IL-2, IL-6 and IL-10. This new Cap-Ab/Det-SOMAmer-based Simoa assay paves the way to develop immunoassays for target proteins where secondary antibodies are not available or have poor binding efficiency. SOMAmer reagents can be selected to resolve the challenges currently encountered with primary and/or secondary antibodies. Future work is needed to demonstrate that Cap-Abs can also be replaced with SOMAmers, thereby fully replacing the antibody pair with a SOMAmer pair. Although SOMAmer pairs are not yet commercially available, this work demonstrates that a SOMAmer-based Simoa assay platform should have great potential for clinical applications of cytokine detection with higher reliability and reproducibility, eventually assisting in the fields of biomarker discovery and disease diagnostics.
Supporting Information Experimental details and supplemental figures/tables. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author * Phone/fax: 617-627-3470 /617-627-3443. Email:
[email protected] Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.
Notes DRW is the scientific founder and a board member of Quanterix, Corp. EK and EO are employees of SomaLogic Inc. and hold equity options in the company. Other authors declare no competing financial interests.
ACKNOWLEDGMENT This work was sponsored by the Defense Advanced Research Projects Agency (agreement # HR0011-12-2-0001; University of North Carolina at Chapel Hill subcontract #5055065). The content of the information does not necessarily reflect the position or the policy of the Government and no official endorsement should be inferred. Approved for public release; distribution is unlimited. Authors thank Somalogic staff, in particular members of SOMAmer Discovery, Chemistry and Analytical Chemistry teams, for selection, synthesis and characterization of SOMAmer reagents used in this work.
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