Digital ELISA is one of the most exciting recent innovations in immunoassay development, leading to demonstrated improvements in sensitivity and low-concentration precision by several orders of magnitude across a broad range of analytes. The principles, theory, methodology and instrumentation are clearly described. The importance and derivation of the average number of enzyme-labeled protein molecules per bead (AEB) are explained. The theory relating to analytical sensitivity and dynamic range is described, with experimental analysis of the performance of the digital ELISA methodological elements. The fundamental limitations of the technique are reviewed, along with possible approaches to overcome them. There is then a step-by-step theoretical analysis of each stage of the assay (analyte capture onto the antibody-coated beads, labeling of the captured analyte with biotinylated detection antibodies, labeling of the biotinylated detection antibodies with enzyme conjugate, and detection of the enzymes). Background and specificity are also reviewed. Assay development is then described for reagents, assay optimization, calibration, and background and interference minimization. Actual assay performance from Digital ELISA assays for several analytes is presented, demonstrating dose-response linearity, sensitivity, reproducibility and accuracy. A number of potential fields that will benefit from the exceptional performance of this technique are reviewed.

The digital ELISA technology described in our chapter has had an eventful time since the publication of the Handbook.  In late 2013, we at Quanterix Corporation launched a fully-automated digital ELISA instrument—shown in the Instrumentation chapter and called the Simoa HD-1 Analyzer—into the research use only (RUO) market.  Since then, the technology has seen rapid adoption by researchers across a number of therapeutic areas and biomedical segments.  Instruments can now be found in a half-dozen countries in university labs, government organizations, pharmaceutical companies, private laboratories offering lab developed tests (LDTs), and contract research organizations (CROs).  These early adopters have been attracted to the unique combination of ultrasensitive immunoassays (typically 1000-fold more sensitive than ELISA) along with multiplexing capability in a fully automated instrument, three key benefits that have not been available before in one technology.

As well as the progress on instrumentation, there have been several developments in the assays or “content” available for digital ELISA.  To accompany the instrument, Quanterix has so far launched 23 digital ELISAs (http://quanterix.com/products/assay-kits), plus a “homebrew” kit that enables researchers to simply develop their own assays for the system.  We also developed an approach for multiplexing of digital ELISA, enabling the detection of up to 10 proteins simultaneously with single molecule sensitivity (Rissin et al., Lab Chip, 2013, 13, pp. 2902-2911).  The capability of the single molecule array (Simoa) technology has also been expanded beyond proteins by the development of a similar approach for detecting nucleic acids, with sensitivities to DNA comparable to the polymerase chain reaction (PCR) (Song et al., Anal. Chem. 85, 1932-1939 (2013)).

Applications for the Simoa HD-1 Analyzer and menu are proving to be wide-ranging, addressing unmet needs in many major therapeutic areas.  Some of the research areas where the technology has had the greatest degree of uptake are neurology, inflammatory disease, oncology, and cardiology.  In neurology, researchers have, for the first time, been measuring a number of brain biomarkers in plasma that previously required a CSF sample.  In cardiology, digital ELISA is being used to identify markers that could be used to predict or stratify risk factors as a form of early monitoring for disease.  In oncology, the early work Quanterix did to demonstrate that its ultra-sensitive PSA test was a reliable predictive marker of cancer recurrence in patients who had had radical prostatectomies has been followed by researchers exploring similar applications for early detection or monitoring of other cancers using a number of different markers.  Finally, inflammatory markers, especially cytokines, have been extremely popular digital ELISAs.  For the first time in many cases, digital ELISA provides the ability to measure these molecules not just in diseased samples but healthy ones too, opening new avenues of research in a number of pathologies where cytokines play a role.  The last 9 months are just the start, but the first Simoa users are discovering new applications for the unique capabilities of this technology. 

David M. Rissin, PhD, is a Principal Scientist at Quanterix Corporation. Dr. Rissin obtained his doctoral degree from Tufts University, where his thesis work focused on the genesis of Single Molecule Array (Simoa) technology. Dr. Rissin is named as an inventor on the earliest patents describing Simoa, and was the first employee of Quanterix Corporation. Dr. Rissin leads the Technology Integration group at Quanterix whose focus is to optimize processes and reagents for successful incorporation with Simoa technology. Dr. Rissin has 3 patents and more than 15 publications in the fields of multiplexed protein detection, single cell analysis, single enzyme molecule kinetics, and single molecule detection.

David C. Duffy, PhD, is Vice President of Research at Quanterix Corporation. Dr. Duffy joined Quanterix in 2007 and leads the team of scientists developing its Single Molecule Array (Simoa) technology. Dr. Duffy was previously at Surface Logix, Gamera Biosciences, and Unilever. Dr. Duffy was a postdoctoral fellow at Harvard University, and was the first Sir Alan Wilson Research Fellow of Emmanuel College, University of Cambridge. Dr. Duffy obtained his doctoral and bachelor degrees at the University of Cambridge. Dr. Duffy has 16 U.S. patents and more than 30 publications in the fields of surface chemistry, microfluidics, and single molecule detection.

David H. Wilson is Vice President of Product Development at Quanterix Corporation, where he heads development of immunoassay products utilizing Quanterix’s single molecule array (Simoa) technology. Prior to Quanterix, he spent 20 years in immunodiagnostic product development at Abbott Laboratories, where he led immunoassay development programs, companion diagnostics programs, and OEM product development programs. He has authored over 40 articles and abstracts. He received his Ph.D. in biophysical chemistry from the University of Illinois/Chicago.

Digital ELISA, sensitivity, ELISA, limit of detection, microarrays, single molecule array, beads, magnetizeable beads, dynamic range, non-specific binding, background, fluorescence, protein capture, on-rate, kinetics, avidin-biotin, efficiency, specificity, assay optimization, matrix effects, assay interference, dose response, linearity, precision, reproducibility, accuracy, calibration, assay speed, load-seal-image modeule,