The COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1–7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.

The COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. Despite rapid advances in diagnostic test development and scale-up, there remains an ongoing need for SARS-CoV-2 tests which are highly sensitive, specific, minimally invasive, cost-effective and scalable for broad testing and surveillance. Here we report development of a highly sensitive single molecule array (Simoa) immunoassay on the automated HD-X platform for the detection of SARS-CoV-2 Nucleocapsid protein (N-protein) in venous and capillary blood (fingerstick). In pre-pandemic and clinical sample sets, the assay has 100% specificity and 97.4% sensitivity for serum / plasma samples. The limit of detection (LoD) estimated by titration of inactivated SARS-CoV-2 virus is 0.2 pg/ml, corresponding to 0.05 Median Tissue Culture Infectious Dose (TCID50) per ml, > 2000 times more sensitive than current EUA approved antigen tests. No cross-reactivity to other common respiratory viruses, including hCoV229E, hCoVOC43, hCoVNL63, Influenza A or Influenza B, was observed. We detected elevated N-protein concentrations in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals using capillary blood from a finger-stick collection device. The Simoa SARS-CoV-2 N-protein assay has the potential to detect COVID-19 infection via antigen in blood with similar or better performance characteristics of molecular tests, while also enabling at home and point of care sample collection.

Background:High-level expression of the Fcγ receptor, CD32hi, on CD4+ T cells was associated with enhanced human immunodeficiency virus (HIV) infection of the latent reservoir in a study of adults receiving antiretroviral therapy. We tested the hypothesis that CD32 was the preferential marker of the latent HIV reservoir in virally suppressed, perinatally HIV-infected adolescents. Methods:The frequency of CD32hiCD4+ T cells was determined by flow cytometry (N = 5) and the inducible HIV reservoir in both CD32hi and CD32-CD4+ T cells was quantified (N = 4) with a quantitative viral outgrowth assay. Viral outgrowth was measured by the standard p24 enzyme-linked immunosorbent assay and an ultrasensitive p24 assay (Simoa; Quanterix) with lower limits of quantitation. Results:We found a 59.55-fold enrichment in the absolute number of infectious cells in the CD32- population compared with CD32hi cells. Exponential HIV replication occurred exclusively in CD32-CD4+ T cells (mean change, 17.46 pg/mL; P = .04). Induced provirus in CD32hiCD4+ T cells replicated to substantially lower levels, which did not increase significantly over time (mean change, 0.026 pg/mL; P = .23) and were detected only with the Simoa assay. Conclusions:Our data suggests that the latent HIV reservoir resides mainly in CD32-CD4+ T cells in virally suppressed, perinatally HIV-infected adolescents, which has implications for reservoir elimination strategies.

Blood based biomarkers for Alzheimer's disease (AD) are urgently required. Extracellular Tau (eTau), a candidate blood biomarker, is highly heterogeneous and the majority of eTau is C-terminally truncated1. Thus, we devised a series of immunoassays to quantify different forms of eTau. We report that when used in plasma a particular N-terminal tau assay (NTT1) can distinguish patients from controls. CSF and plasma were from discovery and validation cohorts of patients with AD dementia (AD), mild cognitive impairment-AD (AD-MCI), and cognitively normal controls (NC). CSF Aß1-42 and total tau (tau) were measured by a single operator using Innotest ELISA. Patients had an AD CSF profile (Aß1-42≤630 pg/ml, Tau/Aß1-42≥0.882); AD patients had MMSE 15-24/30, and AD-MCI had MMSE 25-29/30. NC had Aß1-42>630 pg/ml, Tau/Aß1-42≤0.52 and MMSE 28-30/30. The NTT1 immunoassay was performed using the SimoaTM platform and employs the anti-tau mAbs, BT2 and Tau12. One-way ANOVA were used to compare mean levels of NTT1-measured analyte between groups. Receiver operating characteristics (ROC) curves quantified the diagnostic ability of the plasma assay. Demographic and CSF characteristics for the discovery (AD=25, AD-MCI=21, NC=19) and validation (AD=23, AD-MCI=22, NC=41) cohorts are in Table 1. The two cohorts were well-matched for MMSE and Aß1-42; the AD patients in the validation cohort were older (p<0.0001) and had lower CSF tau levels (p<0.05) than those in the discovery cohort. Results of the NTT1 assays are shown in Figure 1. In the discovery cohort, the NTT1-measured analytes in both CSF and plasma were significantly higher in AD and AD-MCI compared to NC (Figure 1). In the validation cohort, plasma NTT1-measured analytes were again significantly higher in each patient group compared to controls (Figure 1). ROC analysis showed that plasma NTT1 separates controls from AD-MCI (AUC 0.88) and AD (AUC=0.956) in the discovery cohort; in the validation cohort the AUCs were 0.8 and 0.751 respectively. NTT1-measured analyte in CSF (A) and plasma (B) in the discovery cohort and plasma (C) in the validation cohort. Bars show one standard deviation either side of the mean. *** denotes p<0.001 and ** denotes p<0.01 (one-way ANOVA). Plasma NTT1 achieves good separation of AD and AD-MCI from NC in two independent cohorts, and may be a useful screening blood biomarker for AD pathology. Kanmert et al. 2015 J Neurosci 35(30):10851–10865. Weston et al. 2015 Alzheimers Dement (Amst) 1(4):440-446.

Digital ELISA (Enzyme Linked Immunosorbent Assay) based on single molecule arrays (Simoa) has improved sensitivity of traditional ELISA from picomolar (10−12 M) to femtomolar (10−15 M), increasing the quality and quantity of biomarkers that can be measured for health and disease. Digital ELISA counts signal generated from single immunocomplexes formed on superparamagnetic beads confined in arrays of femtoliter-sized wells in which fluorescent molecules are highly concentrated. We have commercialized digital ELISA in a fully-automated instrument (Simoa HD-1 Analyzer), ideal for use in pharmaceutical companies, drug discovery, clinical research and other areas necessitating full automation and high throughput. We have recently launched the SR-X benchtop reader, with a smaller footprint and more flexible workflow. Operators prepare assays in microtiter plates at the bench in a semi-automated format similar to traditional ELISA, with the notable exception that plates are preserved by drying after assay completion, and can be read immediately or the next day. We have so far compared performance of the following Simoa assays on SR-X to HD-1: Tau; P-Tau 181; Neurofilament-light; AB40, AB42, and a neurology multiplex panel consisting of NF-L, tau, GFAP and UCH-L1. Measured sample levels correlated with R2 values from 0.96 to 1.00, with average LOD and LLOQ within 1.4 and 1.5 fold of HD-1, respectively. Inter-assay precision ranged from 4.0 to 11.2% CV across assays. Operators tested full-plates from start to finish within 1 – 2 hours (1/2 hour hands on time) and a read time of 2 hours (5 minutes hands on time). The semi-automated sample prep workflow of the benchtop SR-X system demonstrated good correlation with the fully-automated instrument measuring in serum, plasma and CSF samples.

P2-098 COMPARISON OF TWO PLATFORMS QUANTITATING FG/ML NEUROLOGICAL BIOMARKERSUSING SINGLEMOLECULE ARRAYS AND DIGITAL ELISA: THE BENCHTOP READER SR-X AND THE FULLYAUTOMATED ANALYZER HD-1 Jeremy Lambert, Lei Chang, Linan Song, Purvish P. Patel, Dandan Shan, Joseph Johnson, David Rissin, Quanterix, Lexington, MA, USA; Quanterix Corporation, Lexington, MA, USA; Quanterix Corporation, Cambridge, MA, USA. Contact e-mail: jlambert@quanterix. com

INTRODUCTION:The tau protein plays a central role in Alzheimer's disease (AD), and there is huge interest in measuring tau in blood and cerebrospinal fluid (CSF).METHODS:We developed a set of immunoassays to measure tau in specimens from humans diagnosed based on current best clinical and CSF biomarker criteria.RESULTS:In CSF, mid-region- and N-terminal-detected tau predominated and rose in disease. In plasma, an N-terminal assay (NT1) detected elevated levels of tau in AD and AD-mild cognitive impairment (MCI). Plasma NT1 measurements separated controls from AD-MCI (area under the curve [AUC] = 0.88) and AD (AUC = 0.96) in a discovery cohort and in a Validation Cohort (with AUCs = 0.79 and 0.75, respectively).DISCUSSION:The forms of tau in CSF and plasma are distinct, but in each specimen type, the levels of certain fragments are increased in AD. Measurement of plasma NT1 tau should be aggressively pursued as a potential blood-based screening test for AD/AD-MCI.

Digital ELISA (Enzyme Linked Immunosorbent Assay) based on single molecule arrays (Simoa) has improved sensitivity of traditional ELISA from picomolar (10−12M) to femtomolar (10−15M), increasing the quality and quantity of biomarkers that can be measured for health and disease. Digital ELISA counts signal generated from single immunocomplexes formed on superparamagnetic beads confined in arrays of femtoliter-sized wells in which fluorescent molecules are highly concentrated. Quanterix has developed digital ELISA assays in a fully-automated instrument (Simoa HD-1 Analyzer), ideal for use in pharmaceutical companies, drug discovery, clinical research and other areas necessitating full automation and high throughput. Recent advancements in the Simoa technology and workflow have been integrated into the new SR-X benchtop reader, with a smaller footprint and more flexible workflow. Operators prepare assays in microtiter plates at the bench in a semi-automated format similar to traditional ELISA, with the notable exception that plates are preserved by drying after assay completion, and can be read immediately or the next day. We present results comparing performance of the following Simoa assays on SR-X to HD-1: PSA; HIV p24; Tau; Neurofilament-light; PD-L1; TNFa; IL-10; IL-17A; IL-6 and a neurology multiplex panel consisting of NF-L, tau, GFAP and UCH-L1. Measured sample levels correlated with R2 values from 0.96 to 1.00, with average LOD and LLOQ within 1.4 and 1.5 fold of HD-1, respectively. Inter-assay precision ranged from 4.0 to 11.2% CV across assays. Operators tested full-plates from start to finish within 1 – 2 hours (1/2 hour hands on time) and a read time of 2 hours (5 minutes hands on time).

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.

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.

BACKGROUND The association between increases in cardiac troponin and adverse cardiac outcomes is well established. There is a growing interest in exploring routine cardiac troponin monitoring as a potential early indicator of adverse heart health trends. Prognostic use of cardiac troponin measurements requires an assay with very high sensitivity and outstanding analytical performance. We report development and preliminary validation of an investigational assay meeting these requirements and demonstrate its applicability to cohorts of healthy individuals and patients with heart failure. METHODS On the basis of single molecule array technology, we developed a 45-min immunoassay for cardiac troponin I (cTnI) for use on a novel, fully automated digital analyzer. We characterized its analytical performance and measured cTnI in healthy individuals and heart failure patients in a preliminary study of assay analytical efficacy. RESULTS The assay exhibited a limit of detection of 0.01 ng/L, a limit of quantification of 0.08 ng/L, and a total CV of 10% at 2.0 ng/L. cTnI concentrations were well above the assay limit of detection for all samples tested, including samples from healthy individuals. cTnI was significantly higher in heart failure patients, and exhibited increasing median and interquartile concentrations with increasing New York Heart Association classification of heart failure severity. CONCLUSIONS The robust 2-log increase in sensitivity relative to contemporary high-sensitivity cardiac troponin immunoassays, combined with full automation, make this assay suitable for exploring cTnI concentrations in cohorts of healthy individuals and for the potential prognostic application of serial cardiac troponin measurements in both apparently healthy and diseased individuals.

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 R2>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.

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.

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.

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 0.008pg/mL and 0.006pg/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.

Amyloid β (Aβ) 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 Aβ accumulation in familial AD, less is known about the cause(s) of brain amyloidosis in sporadic disease. Animal and postmortem studies suggest that Aβ 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 Aβ42 concentration in 25 resuscitated patients with severe hypoxia due to cardiac arrest. After a lag period of 10 or more hours, very clear serum Aβ42 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 Aβ levels in blood. The results point to the possibility that hypoxia may play a role in the amyloidogenic process of AD.

Serum Measurement of Hypoxia-Induced Amyloid Beta 1-42 Following Resuscitation from Cardiac Arrest David Wilson, Cambridge, MA, Erik Mortberg, Sten Rebertsson, Uppsala, Sweden, Kaj Blennow, Gothenburg, Sweden, Henrik Zetterberg, Goteborg, Sweden, Linan Song, Lei Chang, Gail Provuncher, Purvish Patel, Evan Ferrell, David Fournier, Cheuk Kan, Todd Campbell, Andrew Rivnak, Brian Pink, Kaitlin Minnehan, Tomasz Piech, David Rissin, David Duffy, Cambridge, MA BACKGROUND: Amyloid beta (A ) peptides are proteolytic products from amyloid precursor protein (APP). Accumulation of A in the form of extracellular plaques is a hallmark of Alzheimer’s disease (AD). Oxidative stress can increase the production of A , which may potentiate AD onset and vascular dementia. It is believed hypoxic insults trigger A production by activating APP proteolysis via a pathway involving -secretase (BACE1) in which BACE1 is upregulated by the transcription factor hypoxia-inducible factor-1 (HIF-1). Animal models have helped elucidate this pathway, but a direct link between hypoxia and A production in the human brain has not been established. We employed a new technology (Single Molecule Arrays, SiMoA) capable of ultrasensitive protein measurements to look for changes in serum A in patients following cardiac arrest and resuscitation. METHODS: 26 unconscious patients with cardiac arrest were resuscitated with restoration of spontaneous circulation (ROSC). Serial blood samples were collected within 6h after cardiac arrest, and continued at intervals from 1-108h. Inclusion criteria included age, systolic BP 80mmHg after ROSC, and a Glasgow Coma Scale 7. Serum aliquots were frozen until assay. Samples were measured in triplicate by SiMoA A assay, which has a limit of detection of less than 0.04 pg/mL. RESULTS: Nearly all patients exhibited a significant time-dependent elevation of A . After a lag period of 10 or more hours, very clear A elevations were observed in most cases. Elevations ranged from approximately 50% to over 30-fold, with most elevations in the range of 3-10-fold (average approximately 7-fold). CONCLUSIONS: These data are the first to directly link hypoxic stress to Aâ elevation in humans. The kinetic profiles may be related to time-dependence of the HIF-1/BACE1 upregulation pathway leading to a hypoxia-induced amyloid cascade. The relevance of mild chronic ischemia with upregulation of the amyloid cascade in AD pathogenesis remains to be examined. Disclosure: Dr. Wilson has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Mortberg has nothing to disclose. Dr. Rubertsson has nothing to disclose. Dr. Blennow has received personal compensation for activities with Innogenetics, Novartis, Janssen Alzheimer Immunotherapy, and Pfizer, Inc as an advisory board member. Dr. Zetterberg has nothing to disclose. Dr. Song has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Chang has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Provuncher has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Patel has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Ferrell has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Fournier has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Kan has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Campbell has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Rivnak has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Pink has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Minnehan has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Piech has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Rissin has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Duffy has received personal compensation for activities with Quanterix Corporation as an employee. Dr. Duffy holds stock and/or stock options in Quanterix Corporation.

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.

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.