Ultrasensitive Measurements of Multiple Cytokines at the Single Molecule Level

Demonstrated use of SimoaTM technology to measure cyto‐ kines in both singleplex and multiplex configurations with excellent precision and limits of detection in the low fg/mL or sub–fg/mL levels, allowing robust quantification well below what is possible today. Introduction Cytokines serve as central communicators for the immune system. Maintaining the delicate balance in the level of these proteins is essential for overall health. As mediators of the inflammatory response, these small protein molecules freely diffuse in systemic circulation and transmit signals to other cells. This balance is disrupted in many chronic inflam‐ matory diseases, including rheumatoid arthritis, Crohn’s dis‐ ease (CD) and ulcerative colitis. Chronic inflammation has been attributed to the increased production of cytokines that are responsible for triggering inflammation and devel‐ opment of the acute phase response.1 Highly successful therapies for treating chronic inflammatory diseases have been developed that target cytokine pathways and offer sig‐ nificant hope for patients. The use of more disease‐specific, quantitative measures that accurately reflect disease sever‐ ity and patient response to therapy would have several advantages over current methods. The precise and accurate measurement of circulating cyto‐ kines is important for understanding their influence on dis‐ ease pathogenesis, treatment, and prognosis. However, cytokine concentrations in healthy individuals and individu‐ als with chronic inflammatory diseases are often at low sin‐ gle‐digit pg/mL or sub–pg/mL levels.2 As physiological concentrations are close to or below the limit of detection of conventional technologies, not only will many samples go undetected, but also those that are measurable will have inherently high imprecision. As a result, it has proven diffi‐ cult or impossible to accurately distinguish groups of patients or to monitor changes in cytokine concentration after administration of drugs or candidate drugs using assays with pg/mL sensitivity. Cytokine concentrations have, therefore, been of limited diagnostic utility. For the measurement of cytokine concentrations to eventu‐ ally be clinically useful, more sensitive assays are clearly needed. QuanterixTM has developed a novel platform that enables the quantification of proteins present in blood and other body fluids at very low, previously undetectable con‐ centrations. The application of this Single Molecule Array (Simoa) technology will provide medical researchers with an unparalleled tool for detecting low‐abundance biomarkers and help facilitate the development of a new generation of diagnostic products useful for early detection or treatment of disease.3 Unprecedented analytical sensitivity is the key differentiator of Quanterix’s technology, made possible by its proprietary single molecule detection (to learn more about Simoa, see whitepaper 1.0). Study Simoa was used to precisely measure two key cytokines, TNFα and IL‐6, in the blood of patients with CD.4 With fg/mL sensitivity, it was possible to precisely measure these two proteins in the blood of all patients, and to monitor changes in concentration with treatment with approved antibody therapeutics. The analytical performance of these assays was evaluated and used to determine the concentration of these cytokines in patients before and after a 12‐week course of anti‐TNFα therapy (Fig. 1). Plasma TNFα concen‐ trations were reduced by antibody treatment by an average Figure 1 Plot of the concentration of TNFα in the plasma of 9 CD patients before treatment with anti‐TNFα therapy (white bars) and 12 weeks after therapy was initiated (gray bars). With the exception of case 19, patients were anti‐TNFα naive. 1 3 5 11 13 17 19 25 27 Case Number 0.0 1.0 2.0 3.0 4.0 5.0 TN Fα (p g/ m L) Before treatment with anti‐TNFα After treatment with anti‐TNFα Copyright© Quanterix Corp. 2014 Page 1 Ultrasensitive Measurements of Multiple Cytokines at the Single Molecule Level of 48% for the nine patients measured in this study. Simi‐ larly, plasma IL‐6 concentrations were reduced during ther‐ apy in the majority of patients tested. Previous studies have been unable to demonstrate a clear reduction in cytokine levels in blood because of insufficient assay sensitivity. Fur‐ thermore, TNFα levels in CD patients and healthy individuals were below the limits of detection of the most commonly used assays. The improved sensitivity and precision of digital ELISA will enable useful measurements of individual cytokines in blood. However, the ability to minimize sample volumes, to eliminate the need to run multiple assays, and to precisely measure multiple proteins simultaneously is important in several fields, including clinical diagnostics. To this end, mul‐ tiplexed Simoa is being used to measure the concentrations of up to 10 low‐abundance proteins simultaneously. Figure 2 illustrates a 4‐plex cytokine assay for TNFα, IL‐6, IL‐1α, and IL‐1B that is capable of maintaining sensitivity comparable to individual singleplex assays down to low fg/mL detection limits.5 The corresponding protein measurements from nor‐ mal healthy individuals were also measured at levels below the detection limits of conventional ELISA‐based technology (Fig. 3). ConclusionSimoa has the potential to markedly improve our under‐standing of inflammatory diseases, including CD. Quanterixis developing a broad menu of individual and multiplexedcytokine assays to address unmet needs in the life scienceresearch market, including IL‐2, IL‐4, IL‐5, IL‐6, IL‐8, IL‐13, IL‐17a, IFNγ, and TNFα. For a complete listing of availableassays, please visit www.quanterix.com. References1 Cellier C, Sahmoud T, Froguel E, et al. Correlationsbetween clinical activity, endoscopic severity, andbiological parameters in colonic or ileocolonic Crohn'sdisease. A prospective multicentre study of 121 cases.The Groupe d'Etudes Thérapeutiques des AffectionsInflammatoires Digestives. Gut. 1994; 35: 231.2 Reinisch W, Gasché C, Tillinger W, et al. Clinical relevanceof serum interleukin‐6 in Crohn's disease: Single pointmeasurements, therapy monitoring, and prediction ofclinical relapse. Am J Gastroenterol. 1994: 2156.3 Rissin DM, Kan CW, Campbell TG, et al. Single‐moleculeenzyme‐linked immunosorbent assay detects serumproteins at subfemtomolar concentrations. Nat Biotech.2010; 28:595.4 Song L, Hanlon DW, Chang L, et al. Single moleculemeasurements of tumor necrosis factor a andinterleukin‐6 in the plasma of patients with Crohn'sdisease. J Immunol Methods. 2011; 372: 177.5 Rissin DW, Kan CW, Song L, et al. Multiplexed singlemolecule immunoassays. Lab Chip. 2013: 1‐10. Quanterix Whitepaper 4.0Page 2Figure 2 Plots of AEB against protein concentration for four cytokinesmeasured in bovine serum samples spiked with all four cytokines. AEB isthe Simoa unit of measurement that is used to calculate concentration.Figure 3 Fifteen normal serum samples measuredusing the Simoa 4‐plex assay.TNFα (pg/mL)0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 1 10 1000.0110