Name: measuring interleukin-6 in human serum from children with a pediatric concussion using a single molecule counting platform
Uploaded: 2019-08-16T12:36:02
Description: Efforts to expand the precision science knowledge base and promote translation of findings to clinical care remain an important area of ongoing inquiry. ...

Additional support for this project came from Dr. Nicole Osier&#8217;s Rising STARs Award from the University of Texas System Permanent University Fund Bond, as well as the CHPR Grant awarded by the St. David&#8217;s Center for Health Promotion and Disease Prevention Research in Underserved PopulationsOpen access publication costs were generously industry-sponsored by Millipore Sigma.

All the procedures including human subjects described in this protocol have been approved by the Institutional Review Board (IRB) at the University of Texas at Austin. This protocol is specifically adjusted for targeted assay of IL-6 in serum; adjustments to the protocol should be made when assaying other protein biomarkers, consistent with manufacturer&#8217;s instructions.
1. Collect, process, and store samples
<ol>
	<li>Collect blood and process the biological samples for serum according ...

<ol>
	<li>Chase, K. A., Cone, J. J., Rosen, C., Sharma, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+value+of+interleukin+6+as+a+peripheral+diagnostic+marker+in+schizophrenia.">The value of interleukin 6 as a peripheral diagnostic marker in schizophrenia.</a> BMC Psychiatry. 16, 152 (2016).</li><li>Lanquillon, S., Krieg, J. C., Bening-Abu-Shach, U., Vedder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+Production+and+Treatment+Response+in+Major+Depressive+Disorder.">Cytokine Production and Treatment Response in Major Depressive Disorder.</a> Neuropsychopharmacology. 22 (4), 370-379 (2000).</li><li>Mastrokolias, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease+biomarker+progression+profile+identified+by+transcriptome+sequencing+in+peripheral+blood.">Huntington's disease biomarker progression profile identified by transcriptome sequencing in peripheral blood.</a> European Journal of Human Genetics. 23 (10), 1349-1356 (2015).</li><li>Imagawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+sensitive+ELISA+for+human+leptin,+using+monoclonal+antibodies.">Development of a sensitive ELISA for human leptin, using monoclonal antibodies.</a> Clinical Chemistry. 44 (10), 2165-2171 (1998).</li><li>Kurien, B. T., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+review+of+other+notable+protein+detection+methods+on+blots.">A brief review of other notable protein detection methods on blots.</a> Methods in Molecular Biology. 536, 557-571 (2009).</li><li>Garcia, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+sensitivity+and+frequent+cross-reactions+in+commercially+available+antibody-detection+ELISA+assays+for+Taenia+solium+cysticercosis.">Low sensitivity and frequent cross-reactions in commercially available antibody-detection ELISA assays for Taenia solium cysticercosis.</a> Tropical Medicine &amp; International Health. , (2017).</li><li>Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W., Gygi, S. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+quantitation+of+C-reactive+protein+using+labeled+tryptic+peptides.">Mass spectrometric quantitation of C-reactive protein using labeled tryptic peptides.</a> Analytical Biochemistry. 354 (2), 175-181 (2006).</li><li>Link, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta+1+suppresses+autoantigen-induced+expression+of+pro-inflammatory+cytokines+but+not+of+interleukin-10+in+multiple+sclerosis+and+myasthenia+gravis.">Transforming growth factor-beta 1 suppresses autoantigen-induced expression of pro-inflammatory cytokines but not of interleukin-10 in multiple sclerosis and myasthenia gravis.</a> Journal of Neuroimmunology. 58 (1), 21-35 (1995).</li><li>Gaye, B., Sikkema, D., Lee, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+ultra-sensitive+single+molecule+counting+assay+for+the+detection+of+interleukin-13+as+a+marker+for+asthmatic+severity.">Development of an ultra-sensitive single molecule counting assay for the detection of interleukin-13 as a marker for asthmatic severity.</a> Journal of immunological methods. 426, 82-85 (2015).</li><li>Li, W., Jiang, W., Dai, S., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Detection+of+Cytokines+Based+on+Dual+Bar-Code+Strategy+and+Single-Molecule+Counting.">Multiplexed Detection of Cytokines Based on Dual Bar-Code Strategy and Single-Molecule Counting.</a> Analytical Chemistry. 88 (3), 1578-1584 (2016).</li><li>Fodale, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+Ultrasensitive+Mutant+Huntingtin+Detection+in+Human+Cerebrospinal+Fluid+by+Single+Molecule+Counting+Immunoassay.">Validation of Ultrasensitive Mutant Huntingtin Detection in Human Cerebrospinal Fluid by Single Molecule Counting Immunoassay.</a> Journal of Huntington's Disease. , (2017).</li><li>Miao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+utility+of+single+molecule+counting+technology+for+quantification+of+KIM-1+in+patients+with+heart+failure+and+chronic+kidney+disease.">Clinical utility of single molecule counting technology for quantification of KIM-1 in patients with heart failure and chronic kidney disease.</a> Clinical Biochemistry. 50 (16-17), 889-895 (2017).</li><li>Grove, H., Faergestad, E. M., Hollung, K., Martens, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+dynamic+range+of+protein+quantification+in+silver-stained+gels+by+modelling+gel+images+over+time.">Improved dynamic range of protein quantification in silver-stained gels by modelling gel images over time.</a> Electrophoresis. 30 (11), 1856-1862 (2009).</li><li>Sandberg, A., Buschmann, V., Kapusta, P., Erdmann, R., Wheelock, &. #. 1. 9. 7. ;. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Time-Resolved+Fluorescence+To+Improve+Sensitivity+and+Dynamic+Range+of+Gel-Based+Proteomics.">Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics.</a> Analytical Chemistry. 88 (6), 3067-3074 (2016).</li><li>Verch, T., Roselle, C., Shank-Retzlaff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+dilution+error+in+ELISAs+using+an+internal+standard.">Reduction of dilution error in ELISAs using an internal standard.</a> Bioanalysis. 8 (14), 1451-1464 (2016).</li><li>Higgins, K. M., Davidian, M., Chew, G., Burge, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+serial+dilution+error+on+calibration+inference+in+immunoassay.">The effect of serial dilution error on calibration inference in immunoassay.</a> Biometrics. 54 (1), 19-32 (1998).</li><li>Rau, M. E., Ahmed, S. S., Lewis, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+entomophilic+digenean+Plagiorchis+noblei+(Trematoda:+Plagiorchiidae)+on+the+survival+of+Aedes+provocans+under+field+conditions.">Impact of the entomophilic digenean Plagiorchis noblei (Trematoda: Plagiorchiidae) on the survival of Aedes provocans under field conditions.</a> Journal of the American Mosquito Control Association. 7 (2), 194-197 (1991).</li><li>Jani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+Use+and+Fit-for-Purpose+Validation+of+Biomarker+Multiplex+Ligand+Binding+Assays+in+Drug+Development.">Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development.</a> The AAPS Journal. 18 (1), 1-14 (2016).</li><li>Kaisar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+degradome+affected+by+variable+storage+of+human+blood.">Plasma degradome affected by variable storage of human blood.</a> Clinical Proteomics. 13, 26 (2016).</li><li>Kaisar, M., van Dullemen, L. F. A., Th&#233;z&#233;nas, M. L., Charles, P. D., Ploeg, R. J., Kessler, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+Biomarker+Profile+Alterations+during+Variable+Blood+Storage.">Plasma Biomarker Profile Alterations during Variable Blood Storage.</a> Clinical Chemistry. 62 (9), 1272-1274 (2016).</li><li>Decker, M. L., Grobusch, M. P., Ritz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Age+and+Other+Factors+on+Cytokine+Expression+Profiles+in+Healthy+Children-A+Systematic+Review.">Influence of Age and Other Factors on Cytokine Expression Profiles in Healthy Children-A Systematic Review.</a> Frontiers in Pediatrics. 5, 255 (2017).</li></ol>

The use of several specific types of equipment can serve to further simplify the protocol and improve results. A rotisserie rotator can be used to promote adequate suspension of the beads and to save time and energy associated with manual inversion. By using a filtration plate over a standard 96 well plate during centrifugation of the samples, the number of plate transfers can be reduced, further saving time. When performing serial dilution of the standards, a 12-channel pipette and 12-well trough could be used to transf...

The benefits of precision care initiatives are becoming increasingly appreciated, and efforts to expand current clinical applications are underway. This increased interest is fueling inquiry into protein biomarkers that have the potential to provide important insights into physiological processes inside the body that can inform clinical care. Current applications for protein biomarkers include diagnosing conditions1, selecting appropriate therapies2, and tracking response to treatment2. These and other precision care successes have led to an expansion in the number and nature of biomarkers being explored. Many putative biomarkers of interest naturally exist at levels below the limit of detection of standard methods for assaying proteins. Likewise, the relatively slow translation of biomarkers to clinical care and large number of candidate biomarkers in the validation pipeline highlights the need for improved throughput of technologies used for biomarker analysis3. These deficiencies have motivated efforts to develop new methodologies that offer ultra-sensitivity, improved workflow, and other advantages over traditional methods. The purpose of this article is to provide a protocol for using a bead-based single molecule counting technology to measure IL-6 in serum.
Traditional protein quantification methods such as western blot, enzyme-linked immunosorbent assay (ELISA), liquid chromatography, mass spectroscopy, and proprietary polymerase chain reaction (PCR) assays , are characterized by sensitivities in the nanogram (ng; 10-9) or picogram (pg; 10-12) per milliliter (mL) range, restricting detection of proteins at or below the femtogram (fg; 10-15) per mL range4,5,6,7,8. However, many proteins that may provide useful windows into important physiological processes (e.g., cytokine signaling) are low-abundance with endogenous levels falling below the detection limit of conventional protein analysis techniques9,10. Emerging new platforms are improving sensitivity through the development of single molecule counting technologies11,12,13. Ultrasensitive systems facilitate reduced signal-to-noise ratios and, most importantly, detection of concentrations as low as the fg/mL range. The platform is unique from other systems with respect to how it counts the single molecules. During the proprietary elution step, the fluorescent dye-labeled detection antibodies are dissociated from the immunocomplex, resulting in free-floating (i.e., in suspension) fluorochromes. A rotating laser is focused through a high-numeric aperture objective to a diameter less than 4 &#181;m. Emitted light is captured by the same objective then passed through a series of positioned mirrors and a long-pass dichroic filter. The image is captured using a charge-coupled device (CCD) camera and individual photons are counted by an Avalanche Photodiode (APD). All digital events, defined as six standard deviations above a background threshold, are counted using a digital event algorithm across a spectrum of time series for a given standard curve; the ultimate result is highly sensitive and reproducible biomarker quantification. Other technologies exist that that allow for ultrasensitive protein quantification. The first ultrasensitive system on the market was the predecessor to the system described in this manuscript; it is a flow cytometry-based instrument that detects Alex 647-labeled detection antibody-analyte complexes in solution. The solution is aspirated into the instrument and is irradiated by a red laser that is focused through a confocal lens. Read times are more than 11 hours per 384-well plate, due to the time-consuming nature of the aspiration/detection method. Another company then launched fully automated system capable of detecting in the fg/mL. Despite the automation and perceived ease-of-use, the monthly cost of maintenance and initial procurement cost may be prohibitive, especially in academic laboratories.
The digital algorithm described above also addresses the limitation of many instruments with respect to dynamic range, especially for high concentrations14,15. A consequence of the narrow dynamic range characteristic of conventional ELISA and other common protein quantification platforms is the reduced utility for certain clinical populations and/or a need for special preparation (e.g., multiple sample dilutions) for cases and controls. Eliminating variable sample dilutions both reduces employee workload and minimizes the effects of dilution as a potential source of error16,17,18.
The device itself is relatively small (16&#8221; high x 14&#8221; wide x 17.5&#8221; deep / ~55 pounds) and does not require fluidics; these features make the system portable and easy to maintain. The system&#8217;s workflow is designed to improve the speed of data acquisition through automation, options to save templates, and faster sample reads via use of early-termination technology. The typical time to prepare the assay (including incubations) is approximately 3.5 h for the IL-6 kit, but may be longer for other proteins if overnight incubation is recommended. The read time is approximately 1-3 h for a 384 well plate (vs. up to 13 h for other platforms); the exact time of the assay depends on the concentration of the analyte of interest with faster reads occurring at higher concentrations. The system is able to run both bead-based and plate-based assays and sample volume requirements are as low as 5 &#956;L (average of 10-25 &#956;L for pre-clinical samples and 10-100&#956;L for clinical samples). If desired, the plates can be re-assayed, because the dye that is eluted off the immunocomplex is stable if stored at 4 &#176;C. Moreover, the system offers the option to use an optimized serum matrix to produce a standard curve that more closely simulates native levels of the analyte in serum/plasma. The inclusion of optimized serum matrix is unique and is in agreement with guidelines for immunoassays outlined by the American Association of Pharmaceutical Sciences19.
The system&#8217;s primary limitation is that debris/bubbles in the sample or scratches/damage to the underside of the plate can interfere with signal acquisition and adversely affect results, due to the ultrasensitive nature of the instrument. However, when a clean workspace is maintained and care in preparation of samples and handling of plates is exercised, extremely low levels of proteins can be precisely, accurately, and reproducibly quantified. Likewise, other manageable limitations common to all protein quantification methods (e.g., effects of pre-analysis storage20,21) apply; best laboratory practices and manufacturer instructions should be strictly adhered to.
This article is the first to demonstrate this single molecule counting system. A large and growing number of high-quality off-the-shelf and custom kits are available for this platform. Rigorous quality control testing ensures assays are consistent with standards set forth by the World Health Organization. Because this method can be used for a wide variety of sample types and sources (e.g. species), the methods described below are applicable to a number of other clinical populations and animal models. The protocol assays interleukin-6 (IL-6) in human serum collected from a sample of pediatric concussion patients.

<table>
	<tbody>
		<tr>
			<td>1L Stericup Filter (0.22 &#956;m; polyethersulfone)</td>
			<td>MilliporeSigma</td>
			<td>SCGPU11RE</td>
			<td>Optional and not used in this study; used to sterilize remaining 1x wash buffer for storage (up to 1 month).</td>
		</tr>
		<tr>
			<td>384-well SensoPlate</td>
			<td>Griener BioScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL graduated cylinder</td>
			<td>Any</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Any</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well V-bottom polypropylene plate (500 &#956;L)</td>
			<td>Axygen</td>
			<td>781892</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge with plate rotator</td>
			<td>Any</td>
			<td>Varies</td>
			<td>Must be capable of reaching a speed of 1,100 x g</td>
		</tr>
		<tr>
			<td>Container capable of holding 500 mL</td>
			<td>Any</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>De-ionized or disitilled water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Erenna 10x System/Wash Buffer</td>
			<td>MilliporeSigma</td>
			<td>02-0111-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Jitterbug Plate Shaker</td>
			<td>Boekel Scientific</td>
			<td>70-0009-00</td>
			<td>A suitable alternative can also be used.</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Any</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Any</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>Modified microplate washer</td>
			<td>BioTek</td>
			<td>95-0004-05</td>
			<td>The microplate washer is modified to include a sphere mag-plate assembly. A suitable alternative (automated or manual) can be used provided it can be modified to include the mag-plate assembly and also has a vacuum regulator as well as a dispense/waste system including a vacuum pump</td>
		</tr>
		<tr>
			<td>MultiScreenHTS BV 96-well Filter Plate</td>
			<td>EMD millipore</td>
			<td>P-96-450V-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Personal protection equipment</td>
			<td>Any</td>
			<td>Varies</td>
			<td>Gloves, labcoat, closed-toed shoes</td>
		</tr>
		<tr>
			<td>Reservoirs for 12-channel pipettors</td>
			<td>Any</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotisserie Rotator</td>
			<td>Any</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealing tape</td>
			<td>Any</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel pipettes</td>
			<td>Any</td>
			<td>Varies</td>
			<td>Must have pipettes capable of transferring 10-250 &#956;L</td>
		</tr>
		<tr>
			<td>SMC High Sensitivity Human IL-6 Immunoassay Kit</td>
			<td>MilliporeSigma</td>
			<td>03-0089-01</td>
			<td>Human assay optimized for use with serum and EDTA plasma samples. The kit contains the following 9 items (1) Assay Buffer; (2) Beads; (3) Standard Diluent; (4) Detection Antibody; (5) Standard; (6) Control 1; (7) Control 2; (8) Control 3; (9) 10X Wash Buffer; (10) Buffer D; (11) Elution Buffer B.</td>
		</tr>
		<tr>
			<td>SMCxPROTM Complete System</td>
			<td>MilliporeSigma</td>
			<td>95-0100-00</td>
			<td>This is the device used to run the assays and quantify the biomarker; the complete kit comes with the device and the associated computer and software.</td>
		</tr>
		<tr>
			<td>Syringe filter (0.2 &#956;m)</td>
			<td>Any</td>
			<td>Varies</td>
			<td></td>
		</tr>
		<tr>
			<td>Titer Plate Shaker</td>
			<td>VWR</td>
			<td>12620-926</td>
			<td>Optional and not used in this study; used when incubating overnight</td>
		</tr>
		<tr>
			<td>Universal plate cover</td>
			<td>Any</td>
			<td>Varies</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring interleukin-6 in human serum from children with a pediatric concussion using a single molecule counting platform

Efforts to expand the precision science knowledge base and promote translation of findings to clinical care remain an important area of ongoing inquiry. Part of this effort includes quantification of proteins that can be used as biological markers (i.e., biomarkers) of pathophysiological processes and other important cellular and tissue activities. Potential applications for biological markers include disease or injury diagnosis, symptom prognosis, and therapy selection/evaluation. The increased understanding of the current and future utility of protein biomarkers, combined with the realization that many biomarkers normally exist in very low levels, has prompted efforts to develop new protein quantification systems with enhanced sensitivity, improved workflows, and shorter read times. Here, we provide an overview of an ultrasensitive immunoassay system and compatible interleukin-6 (IL-6) assays. This assay platform is bead-based, like many other commercially available systems; however, rather than quantifying the fluorescent signal in the spectral address of bead regions, the ultrasensitive immunoassay system quantifies free-floating fluorochromes using a rotating laser, charge-coupled device (CCD) camera, and avalanche photodiode (APD). This high-performance system can be used to quantify a myriad of protein biomarkers, in a variety of biological specimens collected from many species. In this article, quantification of IL-6 in human serum obtained from pediatric brain injury patients will be used as an exemplar.

Using a Five Parameter Logistic Curve Fit (5PL) for the standard curve generated, the Lower Limit of Quantification (LLoQ) was 1.852 pg/mL and the Limit of Detection (LoD) was 0.011 pg/mL with a 0.996 R squared coefficient. Representative data from the ultrasensitive immunoassay system exported CSV file on the n=17 samples ran can be seen in Supplemental Table 1. For this run, the mean concentrations and coefficient of variation were utilized for direct comparison as seen in Table 2. <st...

Methods are described for ultrasensitive quantification of biomarker proteins using an immunoassay system capable of single molecule counting. The platform can be used to quantify a wide array of proteins in a variety of tissues from several species. This exemplar assays interleukin-6 in human serum from pediatric concussion patients.

Measuring Interleukin-6 in Human Serum from Children with a Pediatric Concussion using a Single Molecule Counting Platform

Efforts to expand the precision science knowledge base and promote translation of findings to clinical care remain an important area of ongoing inquiry. ...