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We have developed an ultrasensitive multiplexed immunoassay using 384-well microtiter plates capable of detecting proteins at subfemtomolar concentrations that requires as little as 2.5 μL of sample. Arrays of up to 4 capture antibodies were patterned on the bottom of the wells of a 384-well plate either by directly printing the capture antibodies or by printing anti-peptide tag anchor antibodies and incubating these arrays with capture antibodies conjugated to the corresponding peptide tags ("customized" assays). Samples were incubated with the antibody arrays and shaken orbitally at 2000 rpm to achieve the greatest sensitivity. Chemiluminescence (CL) from immunocomplexes labeled with horseradish peroxidase was imaged across the entire plate to quantify the amount of protein bound to each antibody spot of the arrays. The 384-well assay had a throughput 5-fold greater than 96-well plates that was achieved from simultaneous imaging of CL in all 384-wells and the use of automated pipettors to allow parallel processing of 384 assays. We developed 4 assays based on the 384-well CL ELISA: a direct print assay for IL-10 (limit of detection (LOD) = 0.075 fM); a customized assay for IL-6 (0.22 fM); a customized pharmacokinetic (PK) assay for measuring adalimumab (7.3 pg/mL); and a customized 4-plex assay for IL-5 (0.1 fM), IL-6 (0.52 fM), IL-10 (0.2 fM), and TNF-α (3.2 fM). The sensitivity and precision of the cytokine assays were comparable to current ultrasensitive protein detection methods in 96-well formats. The PK assay for adalimumab was 650 times more sensitive than a commercially available 96-well plate ELISA. We used the 384-well CL ELISAs to measure endogenous levels of the cytokines in the serum and plasma of healthy humans: the mean concentrations and precision were comparable to those from 96-well immunoassays. This 384-well format with subfemtomolar sensitivity will enable ultrasensitive multiplexed immunoassays to be performed with higher throughput and lower sample volumes than currently possible, a particularly important capability for clinical studies in drug development.

Background Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A+B+), but toxin A–negative, toxin B–positive (A−B+) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B. Methods An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and 44 carriers. Isolates were cultured and assessed for in vitro toxin production and in vivo phenotypes (mouse CDI model). Results There were 7 CDI patients and 6 carriers who had stools with detectable toxin A (TcdA, range 23–17 422 pg/mL; 5.6% of samples overall) but toxin B (TcdB) below the clinical detection limit (>B isolates tested in mice, 4 caused diarrhea, and 3 of those 4 caused lethal disease. Ribotyping demonstrated strain diversity. TcdA-predominant samples were also identified at 2 other centers, with similar frequencies (7.5% and 6.8%). Conclusions We report the discovery of clinical pathogenic C. difficile strains that produce high levels of toxin A but minimal or no toxin B. This pattern of toxin production is not rare (>5% of isolates) and is consistently observed in vitro and in vivo in humans and mice. Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis, treatment, and vaccine development.

We have developed a customizable contact printed multiplex immunoassay capable of simultaneously measuring up to five analytes with attomolar sensitivities. This enzyme-linked immunosorbent assay (ELISA) was based on spotting different antibodies in a circular pattern at the bottom of a microtiter plate well. Unlike traditional antibody printing for ELISA that prints a capture antibody specific to a target of interest, in this ELISA we printed unique "anchor" antibodies at the well surface, each having a high affinity for a specific peptide target. By coupling each peptide to a unique assay capture antibody, this array of anchor antibodies enabled a customizable contact printed multiplex immunoassay workflow. As a proof of concept, we developed a 5-plex assay measuring interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 22 (IL-22), and tumor necrosis factor alpha (TNF-α). Measurements of these five analytes in serum and plasma correlated well between the method utilizing the anchor antibodies and peptides and the traditional capture antibody printing approach, with r2 values of 0.99, 0.93, 0.99, 0.96, and 0.75 for IL-5, IL-6, IL-10, IL-22, and TNFα, respectively. This approach makes customizable multiplex ultrasensitive ELISA available to laboratories without access to the precision printing instrumentation and will be useful for antibody screening, custom assay development, biomarker detection, and protein profiling for diagnostic applications.

This paper describes the need for technologies that improve analytical sensitivity to proteins to better define and monitor the progression from heath to disease over the course of an individual's life. These technologies have the potential to allow the early diagnosis of disease, and trigger treatments at the time when they have the greatest opportunity to be effective. We will describe a technology that we have developed for high sensitivity protein detection, namely, single molecule arrays (Simoa). Simoa is based on the capture of protein molecules on magnetic beads, labeling each protein with an enzyme, and counting of single enzyme labels on beads that are isolated in arrays of femtoliter wells. Simoa has enabled the detection of proteins at subfemtomolar concentrations in a variety of biological fluids. We describe the impact of higher sensitivity of proteins using Simoa on: less invasive testing; earlier detection of disease; providing biomarker baseline profiles for healthy individuals; testing of small sample volumes; monitoring of therapeutic efficacy; faster tests; and detection of proteins in complex samples. We also provide a perspective of how new technologies that allow the low-cost manufacture and miniaturization of Simoa could drive the next wave of analytical devices, including wearables.

Circulating microRNAs are biomarkers reported to be stable and translational across species. MicroRNA-122 (miR-122) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). We developed a single molecule, dynamic chemical labeling (DCL) assay to directly detect miR-122 in blood. The DCL assay specifically measured miR-122 directly from 10 μL of serum or plasma without any extraction steps, with a limit of detection of 1.32 pM that enabled the identification of DILI. Testing of 192 human serum samples showed that DCL accurately identified patients at risk of DILI after acetaminophen overdose (area under ROC curve 0.98 (95% CI; 0.96-1), P

We have characterized the sensitivity and kinetics of a multiplex immunoassay system based on detection of chemiluminescence (CL) at arrays of antibodies. This enzyme-linked immunosorbent assay (ELISA) was based on the spotting of different antibodies in a circular pattern at the bottom of a well of a microtiter plate. Sandwich immunocomplexes within each spot were labeled with horse radish peroxidase, and CL was generated locally to each spot in the array from turnover of luminol substrate. CL from the arrays across the plate was collected in single images; long exposure times were used to maximize sensitivity, and short exposure times were used to extend the dynamic range at higher signals. Image analysis was used to determine the intensity of light from each spot in the array, and intensity was converted to concentration of protein via comparison to a calibration curve. To determine the intrinsic sensitivity of the CL ELISA array, streptavidin horseradish peroxidase (SA-HRP) was captured on an array spotted with biotinylated detection antibodies. The limit of detection (LOD) of SA-HRP was 105 aM, or 3200 enzymes per 50 μL. A single-plex assay for prostate specific antigen (PSA) was developed that had an LOD of 79 aM when the microtiter plate was shaken orbitally, comparable to the most sensitive immunoassays reported to date. Normalization of CL signals in the PSA assay to signal per molecule of SA-HRP showed that the efficiency of the shaken assay was ~40%. When the plates were not shaken, the efficiency was ~4.5%, i.e., ~9-fold lower than when shaken. To better understand the theoretical basis of the sensitivity of these assays, we developed COMSOL numerical models of the binding kinetics at the array for plates that were shaken orbitally and those not shaken. Experimental data from the orbitally shaken PSA assay were best modeled by inertial mixing in a three-layer system that included a 8-μm-thick concentration boundary layer. Experimental data from the unshaken PSA assay were well modeled by diffusion-limited kinetics. A single-plex assay for IL-10 was developed with an LOD of 69 aM or 1.5 fg/mL, and used to measure this cytokine in plasma and serum of 10 healthy individuals. A 5-plex assay for IL-5, IL-6, IL-10, IL-22, and TNF-α was developed with LODs of 56 aM, 237 aM, 69 aM, 88 aM, and 373 aM, respectively. The assay was used to measure these 5 cytokines in the plasma and serum of the same individuals. The correlation in concentration of IL-10 measured in single-plex and multiplex assays was good (r2 = 0.89; bias = 14.5%). The factors that result in the high sensitivity of CL ELISA arrays—mostly high signal to noise ratio of extended chemiluminescent imaging—are discussed. This multiplex CL ELISA could be used for sensitive profiling of multiple proteins for in vitro diagnostics and biomarker detection in the development of therapeutics.

Featured Article: Rissin DM, Kan CW, Campbell TG, Stuart SC, Fournier DR, Song L, et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol 2010;28:595–9.3 Proteins are the functional units of life. Berson et al. opened the field of protein measurements with the development of RIAs in the 1950s (1). In 1971, Engvall and Perlmann reported the ELISA (2) that has since been the staple for protein measurements. In 2010, when our article featured here was published, there were 325 proteins detectable in blood, of which 171 were Food and Drug Administration–approved tests of clinical value (3). Most of these proteins are measured using ELISA. When the human genome was sequenced, it was predicted that there should be nearly 4000 proteins in the human bloodstream (4). Where are the missing proteins? ELISA enabled detection down to picomolar concentrations, but this limit of quantification was insufficient to fully access the human blood proteome. The single-molecule array (Simoa) …

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-1β, 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.

We developed a simple immunoassay capable of differentially detecting toxin B from highly virulent strains of Clostridium difficile (BI/NAP-1/027) in stool. This assay can simultaneously confirm the presence of in vivo toxin production and provide strain-related information relevant to infection control epidemiology and disease prognosis.

We have developed a single probe method for detecting microRNA from human serum using single molecule arrays, with sequence specificity down to a single base, and without the use of amplification by polymerases. An abasic peptide nucleic acid (PNA) probe—containing a reactive amine instead of a nucleotide at a specific position in the sequence—for detecting a microRNA was conjugated to superparamagnetic beads. These beads were incubated with a sample containing microRNA, a biotinylated reactive nucleobase—containing an aldehyde group—that was complementary to the missing base in the probe sequence, and a reducing agent. When a target molecule with an exact match in sequence hybridized to the capture probe, the reactive nucleobase was covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. Single molecules of the biotin-labeled probe were then labeled with streptavidin-β-galactosidase (SβG), the beads were resuspended in a fluorogenic enzyme substrate, loaded into an array of femtoliter wells, and sealed with oil. The array was imaged fluorescently to determine which beads were associated with single enzymes, and the average number of enzymes per bead was determined. The assay had a limit of detection of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay, with target specificity down to a single base mis-match. This assay was used to measure microRNA-122 (miR-122)—an established biomarker of liver toxicity—extracted from the serum of patients who had acute liver injury due to acetaminophen, and control healthy patients. All patients with liver injury had higher levels of miR-122 in their serum compared to controls, and the concentrations measured correlated well with those determined using RT-qPCR. This approach allows rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for clinical use., Quanterix Corporation provided support in the form of salaries and research materials for authors [D.M.R. and D.C.D.]., DestiNA Genomics Ltd. provided support in the form of salaries and research materials for authors [B.L.-L., S.P., and H.I.].

We report a method for the sensitive measurement of genomic DNA based on the direct detection of single molecules of DNA in arrays of femtoliter wells. The method begins by generating short fragments of DNA from large, double-stranded molecules of genomic DNA using either restriction enzymes or sonication. Single-stranded fragments are then generated by melting the duplex, and these fragments are hybridized to complementary biotinylated detection probes and capture probes on paramagnetic beads. The resulting DNA complexes are then labeled with an enzyme (streptavidin-β-galactosidase), and single enzymes associated with these complexes on beads are detected in single molecule arrays (Simoa). DNA concentration is quantified by determining the average number of enzymes per bead via Poisson statistics (digital) or the average bead intensity (analog). The Simoa DNA assay was used to detect genomic DNA purified from S. aureus with an average limit of detection (LOD) of 0.07 fM, or 2100 DNA molecules per 50 μL sample. We used this assay to detect S. aureus spiked into (a) whole blood, with an average LOD of 1100 bacteria per 25 μL sample (0.074 fM), and (b) water from the Charles River, with an LOD of 1300 bacteria per 50 μL sample (0.042 fM). Bacteria were detected in river water without prior purification of DNA. The Simoa DNA assay, which directly detects target DNA molecules without molecular replication, is an attractive alternative to existing sensitive DNA detection technologies that rely on amplification using polymerases, such as the polymerase chain reaction (PCR).

BACKGROUNDMeasurement 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.METHODSWe 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.RESULTSThe assay exhibited a functional sensitivity (20% interassay CV) CONCLUSIONSThe 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.

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.

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.

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 (

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 was1200-fold, with coefficients of variation of10%. The potential of digital immunoassays to advance human diagnostics was illustrated in two clinical areas: traumatic brain injury and early detection of infectious disease.

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.

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.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.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 (P0.0005).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.

CHRISTOPHER A. LEPCZYK,∗ NICO DAUPHINE,† DAVID M. BIRD,‡ SHEILA CONANT,§ ROBERT J. COOPER,∗∗ DAVID C. DUFFY,†† PAMELA JO HATLEY,§§ PETER P. MARRA,∗∗∗ ELIZABETH STONE,††† AND STANLEY A. TEMPLE‡‡§§§ ∗Department of Natural Resources and Environmental Management, University of Hawai‘i at Mānoa, Honolulu, HI 96822, U.S.A., email lepczyk@hawaii.edu †Zoological Society of London, Regent’s Park, London NW1 4RY, United Kingdom ‡Avian Science and Conservation Centre, McGill University, Ste. Anne de Bellevue, Quebec H9X 3V9, Canada §Department of Zoology, University of Hawai‘i at Mānoa, Honolulu, HI 96822, U.S.A. ∗∗Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, U.S.A. ††Pacific Cooperative Studies Unit, Department of Botany, University of Hawai‘i at Mānoa, Honolulu, HI 96822, U.S.A. §§Pamela Jo Hatley, P.A., P. O. Box 47477, Tampa, FL 33646-0113, U.S.A. ∗∗∗Smithsonian Migratory Bird Center, National Zoological Park, Washington D.C. 20008, U.S.A. †††Center for Wildlife Health Research, 24 Goss Road, Pownal, ME 04069, U.S.A. ‡‡Department of Forest and Wildlife Ecology, University of Wisconsin-Madison, Madison, WI 53706, Madison, WI 53706, U.S.A. §§§Gaylord Nelson Institute for Environmental Studies, University of Wisconsin-Madison, Madison, WI 53706, U.S.A.

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-1β 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.

Nymphal Ixodes scapularis Say, the vector of Lyme borreliosis, was most common in forested areas across Shelter Island, Suffolk County, New York, and least common in xeric habitats such as beach and grassland. At the scale of individual house yards, nymphs were most common at wooded edges of property and least common on lawns. The abundance of ticks at yard edges was positively correlated with numbers on lawns and in landscaping, suggesting that tick abundance in woods affects abundances in adjacent yards. Because 57% of all yard area is adjacent to woodlands on Shelter Island, public health efforts to reduce tick populations in wooded areas should supplement efforts by individual yard owners to decrease risk. Methods of tick control for woodlands should also be evaluated for their effect on tick populations in adjacent yard habitats.

Nymphal Ixodes scapularis Say deer ticks were collected at 22 parks or other natural areas on Long Island, New York, to examine the relationship between tick populations and geographic position, size of area, presence of white-tailed deer, Odocoileus virginianus (Zimmerman), and numbers of human Lyme disease cases in adjacent communities. Nymphal ticks were 93% less abundant when deer were absent and were also less common in smaller natural areas. Geographic position on Long Island was not important. Tick numbers were significantly correlated with human Lyme disease incidence in adjacent townships. A second survey of larval ticks from five areas where deer were absent and six where deer were present found larvae present at four of the five sites without deer, but at only 2% of the levels found where deer were present. These results suggest that populations of I. scapularis can occur and reproduce in the absence of white-tailed deer, so that eradication of all deer would greatly reduce, but not eliminate, all risk of Lyme disease.

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.