Cytokines act as main communicators for the immune system. For overall health, it is important to preserve the subtle balance in the level of these proteins.
These small protein molecules, as mediators of the inflammatory response, diffuse freely in systemic circulation and convey signals to other types of cells. However, this subtle balance is disturbed in several chronic inflammatory diseases, such as ulcerative colitis, Crohn’s disease (CD), and rheumatoid arthritis.
Chronic inflammation occurs due to increased production of cytokines that play a role in eliciting inflammation and lead to the acute phase response.1 Highly effective therapies for the treatment of chronic inflammatory diseases have been created that focus on cytokine pathways and provide considerable hope for the affected patients.
The implementation of quantitative and more disease‐specific measures that precisely reflect the extent of the disease and patient response to treatment would have many benefits over existing techniques.
The accurate and precise determination of circulating cytokines is essential for interpreting their impact on disease prognosis, treatment, and pathogenesis. Conversely, the concentrations of cytokines in individuals with chronic inflammatory diseases and in healthy individuals are usually at sub-pg/mL levels or low single‐digit pg/mL.2 Since physiological concentrations are below or close to the limit of detection of traditional technologies, several samples go undetected and those that are measurable tend to have innately high imprecision.
Consequently, it has been demonstrated to be impossible or difficult to precisely differentiate groups of patients or to track changes in cytokine concentration following the administration of drugs or candidate drugs through assays with pg/mL sensitivity. Hence, cytokine concentrations have been associated with reduced diagnostic utility.
As a result, more sensitive assays are undoubtedly required to determine the measurement of cytokine concentrations that would ultimately be clinically useful.
An innovative platform, developed by Quanterix™, helps in quantifying the presence of proteins in various body fluids, including blood, at very low and formerly undetectable concentrations. For medical researchers, the use of this Single Molecule Array (Simoa™) technology will serve as an unprecedented tool for identifying low‐abundance biomarkers and help ease the development of next-generation diagnostic products that would be handy for detecting or treating disease at the early stage.3
The main differentiator of Quanterix™’s technology is unparalleled analytical sensitivity, which is made possible by its patented single-molecule detection.
Simoa™ was used for accurately measuring IL‐6and TNFα—two major cytokines present in the blood of patients suffering from CD.4 Through fg/mL sensitivity, these two proteins in the patients’ blood were precisely determined, and variations in concentration were monitored through treatment with approved antibody therapeutics.
The assays’ analytical performance was assessed and used for measuring the cytokine concentration in patients both before and after a 12‐week course of anti‐TNFα treatment, as indicated in Figure 1.
Figure 1. A plot of the concentration of TNFα in the plasma of nine 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.
Antibody treatment helped in reducing the concentrations of plasma TNFα by an average of 48% for all nine patients determined in this analysis. In the same way, the concentrations of plasma IL‐6 were reduced at the time of the treatment in most of the patients tested.
Earlier studies have not been successful in demonstrating an evident reduction in cytokine levels in the blood due to inadequate assay sensitivity. Moreover, TNFα levels in both healthy individuals and CD patients were observed to be less than the limits of detection of the most frequently used assays.
Thus, the precision of digital ELISA combined with the enhanced sensitivity will help in measuring the presence of individual cytokines in blood. Nevertheless, the potential to reduce sample amounts, to remove the necessity to run numerous assays, and to accurately determine various proteins at the same time is essential in many different domains, including clinical diagnostics.
As such, multiplexed Simoa™ is being utilized to determine the concentrations of around 10 low‐abundance proteins concurrently. A 4‐plex cytokine assay for IL‐1B, IL‐1α, IL‐6, and TNFα is shown in Figure 2, demonstrating how it can maintain sensitivity similar to individual singleplex assays that have as low as fg/mL detection limits.5
Figure 2. Plots of AEB against protein concentration for four cytokines measured in bovine serum samples spiked with all four cytokines. AEB is the Simoa unit of measurement that is used to calculate concentration.
The measurements of corresponding proteins from normal healthy individuals were also determined at levels below the detection limits of traditional ELISA‐based technology, as shown in Figure 3.
Figure 3. Fifteen normal serum samples measured using the Simoa 4-plex assay.
The Simoa™ technology has the ability to evidently improve one’s interpretation of inflammatory diseases, such as CD. Quanterix™ is creating an extensive menu of multiplexed and individual cytokine assays to deal with unmet needs in the life science research field, such as IL-2, IL-4, IL-5, IL-6, IL-8, IL-13, IL-17a, TNFα, and IFNγ.
- Cellier C, Sahmoud T, Froguel E, et al. Correlations between clinical activity, endoscopic severity, and biological parameters in colonic or ileocolonic Crohn’s disease. A prospective multicentre study of 121 cases. The Groupe d’Etudes Thérapeutiques des Affections Inflammatoires Digestives. Gut. 1994; 35: 231.
- Reinisch W, Gasché C, Tillinger W, et al. Clinical relevance of serum interleukin‐6 in Crohn's disease: Single point measurements, therapy monitoring, and prediction of clinical relapse. The American Journal of Gastroenterology. 1994: 2156.
- Rissin DM, Kan CW, Campbell TG, et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nature Biotechnology. 2010; 28: 595.
- Song L, Hanlon DW, Chang L, et al. Single molecule measurements of tumor necrosis factor a and interleukin-6 in the plasma of patients with Crohn’s disease. The Journal of Immunological Methods. 2011; 372: 177.
- Rissin DW, Kan CW, Song L, et al. Multiplexed single molecule immunoassays. Lab Chip. 2013: 1–10.
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