The Ultrasensitive Multiplex Immunodetection of Protein Biomarkers

The potential to measure proteins at much lower concentrations than formerly possible has resulted in new scientific and clinical insights in numerous fields, such as neurology, inflammatory disease, cancer, cardiovascular disease, and infectious diseases.1,2

Digital ELISA from Quanterix

Quanterix has earlier described its method for high-sensitivity immunoassays using Single Molecule Arrays (Simoa), also called digital ELISA.1,3–5 In this method, single protein molecules are trapped on antibody-coated, paramagnetic beads. The trapped proteins are labeled with an enzyme label, and single beads are isolated and sealed in arrays of femtoliter wells exposed to enzyme substrate.

The sealing step limits the fluorescent product of the enzyme-substrate reaction to ~40 fL volume, and within 30 seconds, the fluorescence produced by a single enzyme can be detected using an uncooled CCD camera fitted with a white light excitation source.4

Multiplex detection of biomarkers has materialized as a chief component of clinical measurements in biomedicine, particularly in the pharmacokinetic and pharmacodynamic characterizations of candidate drug therapies.6

Immunoassays with high analytical sensitivity exhibit the capability to provide quantitative data on the clinical condition of all healthy and diseased subjects, and to straightaway quantify the biochemical impact of candidate therapies on the protein target. Multiplex measurements offer the potential to comprehend biochemically complex physiological conditions and responses to those conditions after a drug has been administered, using a single sample.

The restricted availability and usually limited quantities of these pre-clinical or clinical samples render multiplexing appealing from a sample utilization standpoint.

To produce a multiplex digital ELISA on the Simoa platform, paramagnetic beads were labeled using fluorescent dyes of different concentrations and wavelengths, to yield optically unique subpopulations of beads, as shown in Figure 1.

Schematic of the multiplexed digital ELISA process.

Figure 1. Schematic of the multiplexed digital ELISA process.7

Then, antibodies to particular proteins were immobilized to these color-encoded beads. Subsequently, blends of these beads were incubated with a sample, and numerous specific proteins were trapped on their specific beads. These proteins were then labeled using enzymes, suspended in enzyme-substrate, loaded into arrays on the Simoa microfluidic disk, sealed with oil, and imaged fluorescently to establish: (a) the subpopulation and location identity of each bead in the femtoliter wells and (b) the absence or presence of a single enzyme linked with each bead. The images were examined to establish the average enzyme per bead (AEB) for each bead subpopulation.4

These AEB values offer a quantitative parameter for establishing the concentration of each protein.

However, for multiplex and sensitive assays to exert a direct influence on clinical diagnostics and drug development, they must be executed on instrumentation that is dependable, scalable, reproducible, and economical.

Ultrasensitive Multiplex Immunoassays on Simoa HD-1 Analyzer

In a new publication,8 Quanterix showed for the first time the incorporation of these ultrasensitive multiplex immunoassays on a completely automated system, the Simoa HD-1 Analyzer, and illustrated how the system was used to create and test an assay for quantifying six cytokines concurrently, with single-molecule resolution.

The multiplex detection of cytokines is especially vital in comprehending the underlying molecular basis of chronic inflammatory diseases, as well as quantifying the influence of candidate drugs on the physiological concentrations of these molecules. Quanterix demonstrated the utility of the 100% automated six-plex by examining blood samples of patients with Crohn’s Disease (CD) (refer Figure 2) and diabetes.

Shows scatter plots of the concentrations of the six cytokines determined for CD patients before treatment and healthy controls.

Figure 2. Shows scatter plots of the concentrations of the six cytokines determined for CD patients before treatment and healthy controls.


Of the six cytokines, only the mean concentration of IL-6 in CD patients was statistically distinct when compared to healthy controls (p = 0.022). This data is in agreement with the previously reported singleplex evaluation of these samples, where IL-6 displayed a major difference in concentrations, but TNF-α did not.

Comparison of the data from this six-plex assay with the same samples run earlier as singleplex assays (see Figure 3) showed an exceptional correlation between the concentrations established for IL-6 using the two techniques (slope of linear regression = 0.97 and r2 = 0.94).

Plots showing correlation between concentrations of IL-6 in the plasma of CD patients determined using the fully automated, six-plex Simoa assay and the manual, singleplex Simoa assay previously reported.

Figure 3. Plots showing correlation between concentrations of IL-6 in the plasma of CD patients determined using the fully automated, six-plex Simoa assay and the manual, singleplex Simoa assay previously reported.2


In conclusion, the data shown in the latest publication8 suggest that completely automated, multiplexed Simoa assays can facilitate the precise and accurate measurement of cytokine concentrations in clinical samples.

Multivariate tests are turning out to be vital in the diagnosis of complex diseases. These tests are typically based on quantifying numerous nucleic acids using polymerase chain reaction (PCR); therefore, approaches such as multiplexed digital ELISA will significantly expand the potential of these tests by measuring numerous proteins.

Quanterix considers that multiplexed digital ELISA could significantly enhance the quantitative understanding of complicated biological phenomena in cancer, inflammatory diseases, neurology, and infectious diseases.


  1. Rissin DM, Wilson DH, Duffy DC. 2013a. Measurement of single protein molecules using digital ELISA. In: Wild, D. (Ed.), The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques, 4th ed. Elsevier, Oxford, UK.
  2. Song L, Hanlon, DW, Chang L, et al. Single molecule measurements of tumor necrosis factor α and interleukin-6 in the plasma of patients with Crohn’s disease. J Immunol Methods. 2011; 372:177.
  3. Kan CW, Rivnak AJ, Campbell TG, et al. Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies. Lab Chip. 2012; 12:977.
  4. Rissin DM, Kan CW, Campbell TG, et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol. 2010; 28:595.
  5. Rissin DM, Fournier DR, Piech T, et al. Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range. Anal Chem. 2011; 83:2279.
  6. Yu, X., Schneiderhan-Marra, N., Joos, T.O., 2010. Protein microarrays for personalized medicine. Clin. Chem. 56, 376.
  7. Rissin DM, Kan CW, Song L, et al. Multiplexed single molecule immunoassays. Lab Chip. 2013; 13:2902.
  8. Rivnak AJ, Rissin DM, Kan CW, et al., A fully-automated, six-plex single molecule immunoassay for measuring cytokines in blood, J Immunol Methods, 2015; 424:20.

About Quanterix

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Last updated: Feb 1, 2020 at 8:27 PM


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