The Simoa HD-1 Analyzer: A Novel Fully Automated Digital Immunoassay Analyzer with Single-Molecule Sensitivity and Multiplexing.

Disease detection at the molecular level is driving the emerging revolution of early diagnosis and treatment. A challenge facing the field is that protein biomarkers for early diagnosis can be present in very low abundance. The lower limit of detection with conventional immunoassay technology is the upper femtomolar range (10(-13) M). Digital immunoassay technology has improved detection sensitivity three logs, to the attomolar range (10(-16) M). This capability has the potential to open new advances in diagnostics and therapeutics, but such technologies have been relegated to manual procedures that are not well suited for efficient routine use. We describe a new laboratory instrument that provides full automation of single-molecule array (Simoa) technology for digital immunoassays. The instrument is capable of single-molecule sensitivity and multiplexing with short turnaround times and a throughput of 66 samples/h. Singleplex and multiplexed digital immunoassays were developed for 16 proteins of interest in cardiovascular, cancer, infectious disease, neurology, and inflammation research. The average sensitivity improvement of the Simoa immunoassays versus conventional ELISA was >1200-fold, with coefficients of variation of <10%. The potential of digital immunoassays to advance human diagnostics was illustrated in two clinical areas: traumatic brain injury and early detection of infectious disease.

A fully-automated, six-plex single molecule immunoassay for measuring cytokines in blood.

We report a system and assay for performing fully-automated measurement of 6 proteins simultaneously with single molecule sensitivity. The system combines handling of samples, reagents, and consumables, with a module for imaging single molecule arrays (Simoa) to enable immunoassays that have high sensitivity (~fg/mL), are multiplexed, and are fully-automated. A 6-plex cytokine Simoa assay for IL-6, TNF-α, GM-CSF, IL-10, IL-1ß, and IL-1α was developed on the system. The assays had limits of detection in the range 0.01-0.03pg/mL, and the average imprecision (CV) of the Simoa signal was 4.2%. This assay was used to measure the concentrations of these cytokines in the plasma of patients with Crohn's Disease (CD), before and after treatment with anti-TNF-α antibody drugs, and in the serum of Type 1 diabetics. Concentrations of TNF-α and IL-6 in the CD samples determined using the fully-automated, multiplex Simoa assay had good correlation with the manual, single-plex assays previously reported. Drug treatment caused reductions in the mean concentration of all 6 cytokines in the plasma of CD patients. The concentrations of 4 cytokines were significantly higher in diabetics compared to healthy controls. The system could enable the widespread, multiplexed measurement of protein biomarkers with low abundance.

Multiplexed single molecule immunoassays.

We have developed a method that enables the multiplexed detection of proteins based on counting single molecules. Paramagnetic beads were labeled with fluorescent dyes to create optically distinct subpopulations of beads, and antibodies to specific proteins were then immobilized to individual subpopulations. Mixtures of subpopulations of beads were then incubated with a sample, and specific proteins were captured on their specific beads; these proteins were then labeled with enzymes via immunocomplex formation. The beads were suspended in enzyme substrate, loaded into arrays of femtoliter wells--or Single Molecule Arrays (Simoa)--that were integrated into a microfluidic device (the Simoa disc). The wells were then sealed with oil, and imaged fluorescently to determine: a) the location and subpopulation identity of individual beads in the femtoliter wells, and b) the presence or absence of a single enzyme associated with each bead. The images were analyzed to determine the average enzyme per bead (AEB) for each bead subpopulation that provide a quantitative parameter for determining the concentration of each protein. We used this approach to simultaneously detect TNF-α, IL-6, IL-1α, and IL-1ß in human plasma with single molecule resolution at subfemtomolar concentrations, i.e., 200- to 1000-fold more sensitive than current multiplexed immunoassays. The simultaneous, specific, and sensitive measurement of several proteins using multiplexed digital ELISA could enable more reliable diagnoses of disease.

Simple diffusion-constrained immunoassay for p24 protein with the sensitivity of nucleic acid amplification for detecting acute HIV infection.

Nucleic acid amplification techniques have become the mainstay for ultimate sensitivity for detecting low levels of virus, including human immunodeficiency virus (HIV). As a sophisticated technology with relative expensive reagents and instrumentation, adoption of nucleic acid testing (NAT) can be cost inhibited in settings in which access to extreme sensitivity could be clinically advantageous for detection of acute infection. A simple low cost digital immunoassay was developed for the p24 capsid protein of HIV based on trapping enzyme-labeled immunocomplexes in high-density arrays of femtoliter microwells and constraining the diffusion of the enzyme-substrate reaction. The digital immunoassay was evaluated for analytical sensitivity for HIV capsid protein p24, and compared with commercially available NAT methods and immunoassays for p24, including 4th-generation antibody/antigen combo assays, for early detection of HIV in infected individuals. The digital immunoassay was found to exhibit 2000-3000-fold greater analytical sensitivity than conventional immunoassays reactive for p24, and comparable sensitivity to NAT methods. Assaying serial samples from 10 HIV-infected individuals, the digital immunoassay detected acute HIV infection as early as NAT methods, and 7-10 days earlier than conventional immunoassays. Comparison of assay results between the digital immunoassay and a quantitative NAT method from HIV infected serum exhibited a linear correlation R(2)>0.99. The data indicate that by constraining diffusion of the signal generation step of a simple sandwich immunoassay and enabling the digital counting of immunocomplexes, dramatic improvements in sensitivity to virus can be obtained to match the sensitivity of NAT at a fraction of the cost.

Tau proteins in serum predict neurological outcome after hypoxic brain injury from cardiac arrest: results of a pilot study.

OBJECTIVE: To conduct a pilot study to evaluate the prognostic potential of serum tau protein measurements to predict neurological outcome 6 months following resuscitation from cardiac arrest. METHODS: In this retrospective observational study, we employed a new ultra sensitive digital immunoassay technology to examine serial serum samples from 25 cardiac arrest patients to examine tau release into serum as a result of brain hypoxia, and probe for its significance predicting six-month neurological outcome. Serial blood samples were obtained from resuscitated cardiac arrest survivors during their first five days in an intensive care unit, and serum total tau was measured. Cerebral function assessments were made using Cerebral Performance Categorization (CPC) at discharge from the ICU and six months later. Tau data were analyzed in the context of 6-month CPC scores. RESULTS: Tau elevations ranged from modest (<10 pg/mL) to very high (hundreds of pg/mL), and exhibited unexpected bi-modal kinetics in some patients. Early tau elevations appeared within 24h of cardiac arrest, and delayed elevations appeared after 24-48 h. In patients with delayed elevations, areas under the curves of tau concentration vs. hours since cardiac arrest were highly predictive of 6-month outcome (P<0.0005). CONCLUSION: High-sensitivity serum tau measurements combined with an understanding of tau release kinetics could have utility for hypoxic brain injury assessment and prediction of cerebral function outcome.

Single molecule enzyme-linked immunosorbent assays: theoretical considerations.

We have developed a highly sensitive immunoassay-called digital ELISA-that is based on the detection of single enzyme-linked immunocomplexes on beads that are sealed in arrays of femtoliter wells. Digital ELISA was designed to be highly efficient in the capturing of target proteins, labeling of these proteins, and their detection in single molecule arrays (SiMoA); in essence, the goal of the assay is to "capture every molecule, detect every molecule". Here we provide the theoretical basis for the design of this assay derived from simple equations based on bimolecular interactions. Using these equations and knowledge of the concentrations of reagents, the times of interactions, and the on- and off-rates of the molecular interactions for each step of the assay, it is possible to predict the number of immunocomplexes that are formed and detected by SiMoA. The unique ability of SiMoA to count single immunocomplexes and determine an average number of enzymes per bead (AEB), makes it possible to directly compare the number of molecules detected experimentally to those predicted by theory. These predictions compare favorably to experimental data generated for a digital ELISA for prostate specific antigen (PSA). The digital ELISA process is efficient across a range of antibody affinities (K(D)~10(-11) -10(-9) M), and antibodies with high on-rates (k(on)>10(5) M(-1) s(-1)) are predicted to perform best. The high efficiency of digital ELISA and sensitivity of SiMoA to enzyme label also makes it possible to reduce the concentration of labeling reagent, reduce backgrounds, and increasing the specificity of the approach. Strategies for dealing with the dissociation of antibody complexes over time that can affect the signals in an assay are also described.

Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies.

We report a method for isolating individual paramagnetic beads in arrays of femtolitre-sized wells and detecting single enzyme-labeled proteins on these beads using sequential fluid flows in microfabricated polymer array assemblies. Arrays of femtolitre-sized wells were fabricated in cyclic olefin polymer (COP) using injection moulding based on DVD manufacturing. These arrays were bonded to a complementary fluidic structure that was also moulded in COP to create an enclosed device to allow delivery of liquids to the arrays. Enzyme-associated, paramagnetic beads suspended in aqueous solutions of enzyme substrate were delivered fluidically to the array such that one bead per well was loaded by gravity. A fluorocarbon oil was then flowed into the device to remove excess beads from the surface of the array, and to seal and isolate the femtolitre-sized wells containing beads and enzyme substrate. The device was then imaged using standard fluorescence imaging to determine which wells contained single enzyme molecules. The analytical performance of this device as the detector for digital ELISA compared favourably to the standard method, i.e., glass arrays mechanically sealed against a silicone gasket; prostate specific antigen (PSA) could be detected from 0.011 pg mL(-1) up to 100 pg mL(-1). The use of an enclosed fluidic device to isolate beads in single-molecule arrays offers a multitude of advantages for low-cost manufacturing, ease of automation, and instrument development to enable applications in biomarker validation and medical diagnosis.

Hypoxia due to cardiac arrest induces a time-dependent increase in serum amyloid ß levels in humans.

Amyloid ß (Aß) peptides are proteolytic products from amyloid precursor protein (APP) and are thought to play a role in Alzheimer disease (AD) pathogenesis. While much is known about molecular mechanisms underlying cerebral Aß accumulation in familial AD, less is known about the cause(s) of brain amyloidosis in sporadic disease. Animal and postmortem studies suggest that Aß secretion can be up-regulated in response to hypoxia. We employed a new technology (Single Molecule Arrays, SiMoA) capable of ultrasensitive protein measurements and developed a novel assay to look for changes in serum Aß42 concentration in 25 resuscitated patients with severe hypoxia due to cardiac arrest. After a lag period of 10 or more hours, very clear serum Aß42 elevations were observed in all patients. Elevations ranged from approximately 80% to over 70-fold, with most elevations in the range of 3-10-fold (average approximately 7-fold). The magnitude of the increase correlated with clinical outcome. These data provide the first direct evidence in living humans that ischemia acutely increases Aß levels in blood. The results point to the possibility that hypoxia may play a role in the amyloidogenic process of AD.

Fifth-generation digital immunoassay for prostate-specific antigen by single molecule array technology.

BACKGROUND: Measurement of prostate-specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been hindered by the limit of quantification of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, postsurgical PSA is typically undetectable with current assay methods. Evidence suggests, however, that more sensitive determination of PSA status following RP could improve assessment of patient prognosis and response to treatment and better target secondary therapy for those who may benefit most. We developed an investigational digital immunoassay with a limit of quantification 2 logs lower than current ultrasensitive third-generation PSA assays. METHODS: We developed reagents for a bead-based ELISA for use with high-density arrays of femtoliter-volume wells. Anti-PSA capture beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. We characterized analytical performance, compared its accuracy with a commercially available test, and analyzed longitudinal serum samples from a pilot study of 33 RP patients. RESULTS: The assay exhibited a functional sensitivity (20% interassay CV) <0.05 pg/mL, total imprecision <10% from 1 to 50 pg/mL, and excellent agreement with the comparator method. All RP samples were well within the assay measurement capability. PSA concentrations following surgery were found to be predictive of prostate cancer recurrence risk over 5 years. CONCLUSIONS: The robust 2-log improvement in limit of quantification relative to current ultrasensitive assays and the validated analytical performance of the assay allow for accurate assessment of PSA status after RP.

Single molecule measurements of tumor necrosis factor α and interleukin-6 in the plasma of patients with Crohn's disease.

The quantitative measurement of inflammatory cytokines in blood has been limited by insufficient sensitivity of conventional immunoassays. This limitation has prevented the widespread clinical monitoring of cytokine concentrations in chronic inflammatory diseases. We applied a sensitive, single molecule detection technology to measure TNF-α and IL-6 in the plasma of patients with Crohn's disease (CD), before and after treatment with anti-TNF-α therapy. Plasma from 17 patients with CD was collected prior to initiation of anti-TNF-α therapy, and the Crohn's disease activity index (CDAI) was determined for each patient. A sub-set of these patients returned for follow up 12 weeks after treatment started. Plasma from age- and gender-matched controls was also collected. Digital ELISAs were developed for TNF-α and IL-6, and the plasma concentrations of these cytokines were determined using digital ELISA. The limits of detection of the TNF-α and IL-6 digital ELISAs were 0.008 pg/mL and 0.006 pg/mL, respectively. Both cytokines were detected in all samples using digital ELISA and the concentrations of TNF-α and IL-6 in the plasma of patients with CD were (3.6±0.9) pg/mL and (10.9±11.2) pg/mL, respectively. TNF-α levels in patients and healthy controls were not significantly different, but the IL-6 levels in plasma were significantly elevated in patients compared to controls. After therapy, the mean reduction of the concentrations of free TNF-α and IL-6 were 46% and 58%, respectively. Digital ELISA provided the first quantitative measurements of TNF-α and IL-6 concentrations in the plasma of all patients in a population with CD. The changes in cytokine concentrations after therapy--which could be quantified because of the high sensitivity of digital ELISA--could be used for monitoring therapeutic efficacy.

Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range.

We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ∼1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.

Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations.

The ability to detect single protein molecules in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as approximately 10-20 enzyme-labeled complexes in 100 microl of sample (approximately 10(-19) M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (<10(-15) M) much lower than conventional ELISA. Digital ELISA detected prostate-specific antigen (PSA) in sera from patients who had undergone radical prostatectomy at concentrations as low as 14 fg/ml (0.4 fM).