Publications

Background: While numerous new mouse models of diabetes have been developed to study the pathophysiology of this complex disease, very few efforts have been made to improve the methodology for the accurate measurement of insulin in mice. To robustly measure insulin concentrations in small volumes (≤10µL) of mouse plasma, sensitivity, accuracy and precision of immunoassays should definitely be improved. Although ultra-sensitive ELISA has been available that allows us to detect low concentrations of insulin in small volumes of mouse plasma, this method usually comes at the expense of accuracy and precision especially in the low range of insulin concentrations.

Objective: We report a comparative analysis of an ultra-sensitive immunoassay for mouse insulin based upon the single molecule counting technology of the ErennaTM System (Singulex) to two other assays based on the ultra-sensitive ELISA method.

Methods: Mouse insulin concentrations were determined by three different methods: the Erenna System (5µL plasma), the ALPCO mouse ultra-sensitive ELISA (5µL plasma), and the Linco/Millipore ELISA (10µL plasma). Samples were analyzed using a 384-well plate format with monoclonal capture and detection antibodies. Human insulin, which exhibits complete cross-reactivity in these methods, was used as the standard reference. For the Singulex vs. Linco/Millipore comparison, plasma was purchased from starved CD-1, Balb/c and C57BL/6 mice (Innovative Research). Mouse plasma samples (~100µL) for the Singulex vs. ALPCO comparison were provided by the Permutt Laboratory (WUSM).

Results: The Singulex Erenna and the ALPCO methods with 5µL mouse plasma resulted in analytical concentrations of insulin ranging from 503-2786 pg/mL vs. 936-2918 pg/mL, respectively. Linear regression of each data set showed that insulin concentrations determined by these two respective assays were highly correlated, but slight up-shifts of measured insulin concentrations were observed in the ALPCO method (y = 1.02x - 342, R2 = 0.936). Sensitivity, lower limit of quantification, and linearity were 12 pg/mL vs. 115 pg/mL, 20 pg/mL vs. 125 pg/mL, and 20-1280 pg/mL vs. 278-1280 pg/mL between the Erenna and the ALPCO methods, respectively. Within-assay precisions and measured insulin concentrations were 2-4% vs. 5-26% and 150-359 pg/mL vs. 119-326 pg/mL for the Erenna and the ALPCO methods, respectively. We also compared Singulex Erenna assays with 5µL plasma and Linco/Millipore assays with 10µL plasma. Measured insulin levels ranged 94-1961 pg/mL vs. 351-2002 pg/mL, respectively, and the measured values were highly correlated.

Conclusion: The comparison of the novel Singulex ErennaTM System and other ELISA methods clearly showed that the Erenna method had superior precision and accuracy with a smaller sample volume over currently available ELISA methods. Therefore, the single molecule detection technology of the Singulex ErennaTM System is able to meet much higher requirements of insulin measurement particularly when a small sample volume and low analyte concentration are evident, but a high level of precision and accuracy are required. Currently, we are attempting to apply this novel technology to measure insulin concentrations in several different mouse models.

Background: Detection of cardiac injury by measurement of cardiac troponin (cTn) is essential for diagnosis of acute myocardial infarction (AMI) and provides information for risk stratification. Recent guidelines recommend use of the 99th percentile cutoff value with assay imprecision <10%, which most commercial assays do not meet. In addition, cTn values below the 99th% value provide diagnostic and prognostic information. Recently, a novel assay, the ErennaTM cTnI Immunoassay (Singulex) utilizing single photon fluorescence detection and paramagnetic microparticles (MPs) has been shown to have increased sensitivity and precision. However, the analytical specificity of this assay has not been shown.

Objective: Accordingly, we investigated the specificity of the Erenna MP-based cTnI Immunoassay for quantification of cTnI concentration in normal human subjects in matched serum and plasma (three different anticoagulants) samples.

Methods: We collected serum, EDTA, lithium heparin and citrate plasma from 20 random subjects free of overt cardiac disease and symptoms (13 female, 7 male, average 43y, range 23-64y). Concentrations of cTnI were determined in triplicate by the Erenna assay for each specimen type as an average (+/-SDEV). The average cTnI value was assessed across the 4 matched specimen types. To test for specificity, MPs were coated with either a cTnI capture monoclonal antibody (R&D Systems, Minneapolis MN), an unrelated monoclonal antibody directed against the beta-amyloid 42-mer peptide (αA-beta42, Covance) or left un-coated (blank MPs). Precision of the Erenna assay was determined by calculating average concentration and %CV of cTnI from two control sera over 14 assay runs performed over 4 days.

Results: cTnI was quantifiable in all serum and plasma specimens, and no significant differences in cTnI concentrations across samples were observed. No trends were observed in cTnI values related to age or sex. Average cTnI concentration across the 4 specimen types ranged from 0.84-22.37 ng/L, with an average of 3.76 (+/- 4.76) ng/L. Two donors provided significantly elevated average cTnI measurements (9.6 and 22.4 ng/L respectively). Taking the 22.4 ng/L sample as a statistical outlier, removal from the analysis yielded an adjusted average of 2.78 (+/- 1.93) pg/ml, similar to that reported previously. cTnI was undetectable (<0.1 ng/L) in 20/20 donor samples when blank MPs were used as the solid phase capture material and 16/20 samples when the MPs were coated with anti A-beta-42. The remaining 4 donors provided a 50%, 65%, 70%, and 82% reduction in cTnI concentration, respectively, and this non-specific binding accounted for 18-50% of the total cTnI signal. Precision of the Erenna assay using control sera were 7% CV at 8.3 ng/L and 10% CV at 2.2 ng/L.

Conclusion: Our data demonstrate that the Erenna MP-based cTnI Immunoassay has high specificity and can be used to reliably measure cTnI in apparently healthy human subjects. This high degree of specificity and high sensitivity should allow for further investigation of the clinical significance of mild increases in cTnI in patients with and without acute coronary disease.

Many cardiovascular (cTnI), cancer (VEGF, PSA), inflammation (cytokines), and metabolic (insulin, GLP-1) biomarkers used for diagnostics cannot be measured in normal, healthy human subjects using currently available technologies due to extremely low circulating concentrations. However, in order to understand the progression of disease and evaluate effectiveness of therapeutics, it is essential to understand the baseline concentrations of these biomarkers in healthy subjects. To address this problem, Singulex has developed the ErennaTM MP-based Immunoassay System, which currently leads the next generation of molecular diagnostic technologies capable of quantifying biomarkers at the sub-picogram level. The Erenna technology utilizes single-photon fluorescence counting to provide a broad dynamic range and paramagnetic microparticles (MPs) for capture and detection of analyte from a complex biological sample, providing specificity, sensitivity and precision. Through an Erenna Technology Access Program (ETAP), Singulex has fostered key collaborations in both academia and the pharmaceutical industry that have demonstrated the utility and value of the Erenna Immunoassay System for applications defining clinically relevant biology around existing and emerging biomarkers. Cardiac troponin (cTnI) is considered the gold-standard biomarker for diagnosis of acute myocardial infarction (AMI) and cardiotoxicity. The Erenna System has been used to specifically quantify cTnI in serum from healthy human subjects, which is below the limit of detection for the previous generation of immunoassays. Additional studies have investigated the short- and long-term biological variation of cTnI, for use in clinical diagnosis of acute coronary syndrome (ACS) and chronic cardiac disease. The Erenna System can also detect low-level changes in circulating cTnI for subjects experiencing transient myocardial ischemia (TMI). There is also applicability for studies of cardiotoxicity in established animal model systems, and the Erenna System has been used to successfully measure the temporal variability of cTnI in rats. One feature of the Erenna System is the flexibility with which the assay can be customized to meet diverse diagnostic needs of investigators. The Erenna System has been successfully utilized to measure a diverse panel of important biomarkers for human disease. Baseline levels of cytokine concentrations from plasma of normal human subjects have been measured using the Erenna System, previously immeasurable by current assay technologies. Prostate specific antigen (PSA) has been quantified at exceptionally low concentrations both in male and female subjects, with promise for cancer diagnostics and therapeutics. The Erenna System has also been utilized to detect insulin levels in small volumes (≤10µL) of mouse plasma, allowing the next generation of mouse models for diabetes to be evaluated using molecular diagnostics. These collaborative efforts have shown that the Erenna System can be utilized to initiate studies requiring extreme sensitivity, and which were previously untenable. Such improvements in molecular detection technology are essential for developing biomarker baseline profiles for better diagnosis of disease, development of promising new diagnostic information and evaluation of therapeutic interventions.

Background: Cytokines are a complex family of secreted signaling molecules which trigger a variety of responses from the immune system in a combinatorial manner. Thus, the level of secretion of specific cytokines can have implications for a variety of pathologies, including inflammation, cancer, diabetes, and autoimmune disorders, to name a few. Interestingly, baseline concentrations from plasma of normal human subjects for many cytokines have yet to be defined by current assay technologies. Recently, a novel assay, the ErennaTM Immunoassay System (Singulex) utilizing single-photon fluorescence detection and paramagnetic microparticles (MPs) has been shown to have increased sensitivity and precision, making it a good candidate technology for such determinations.

Objective: We investigated the utility of the Erenna MP-based Immunoassay for quantification of a specific panel of cytokine concentrations in normal human plasma samples.

Methods: Human plasma was obtained from normal blood bank donors, with no apparent health concerns. Samples (100µl) were assayed for a panel of cytokines with the Erenna MP-based Immunoassay System (Singulex), which utilized paramagnetic microparticles as the solid phase format in combination with single-molecule counting. The assay panel consisted of: IL-1-beta, IL-6, IL-8, and IL-17 capture and detection antibodies in a sandwich assay format (R&D Systems). In the Erenna assay system, the immunoassay complex formed on the MP surface results in the release of fluorescently labeled detection antibody. The resulting solution is sipped into a 100µl flow capillary and photons are counted, via confocal microscopy, as they pass through a 2µm interrogation space. A large dynamic range of 4+ logs is obtained by combining single-molecule counting (low range) with photon counting (mid range) and total light measurements (high range). The sensitivity of each assay (LoD in pg/mL) and concentration range (pg/mL) for each cytokine in normal plasma was determined.

Results: Normal distributions were observed for each cytokine assayed in the panel: IL-1-beta (n=16), IL-6 (n=32), IL-8 (n=32), IL-17 (n=9). For IL-6 and IL-8, the concentration of each cytokine was quantifiable in all plasma specimens (32/32). For IL-1-beta, average cytokine concentration ranged from 0.05-0.25 pg/mL, with an assay LoD at 0.02 pg/mL (y = 771x + 375, R2=0.99). For IL-6, average cytokine concentration ranged from 0.2-26 pg/mL (Mean 2.3 pg/mL), with an assay LoD at 0.01 pg/mL (y = 1900x + 165, R2=0.99). For IL-8, average cytokine concentration ranged from 1.2-26 pg/mL (Mean 7.3 pg/mL). For IL-17, average cytokine concentration ranged from 0.2-0.7 pg/mL, with an assay LoD at 0.05 pg/mL (y = 0.86x + 5.4, R2=0.999) and LLoQ at 0.1 pg/mL.

Conclusion: Molecular detection technology has a broad range of applications, including clinical diagnostics and pharmaceutical development. The Erenna MP-based Immunoassays have low limits of detection, measuring a variety of low cytokine concentrations in normal human subjects. Such improvements in molecular detection technology are essential to understanding how deviations from normal cytokine secretion levels correspond to development of disease, and whether normal secretion patterns return after therapeutic intervention.

Background: The European Society/American College of Cardiology established cardiac troponin (cTnI) as the gold standard for diagnosis of acute myocardial infarction (AMI) and risk stratification for adverse cardiac events. We previously reported a highly sensitive cTnI assay and the use of this assay to define cTnI levels in normal subjects. This assay has been further developed to yield greater sensitivity and currently we report its analytical and preliminary clinical performance.

Methods: The Singulex cTnI assay was modified to use paramagnetic microparticles (MP) as the solid phase with a monoclonal capture antibody and a fluorescently-tagged affinity-purified goat detection antibody. After incubations and washing, the fluorescently-tagged antibody is chemically released from MPs and an aliquot is pumped into the Erenna™ Digital Molecule Counting (DMC) System. Individually-labeled antibodies are measured during capillary flow by setting the interrogation volume such that the emission of single fluorescent molecules is detected in a defined space following laser excitation. Total fluorescent signal is determined as a sum of the individual digital events. A 4-log dynamic range is obtained by analyzing digital events as well as total photons. The limit of detection the Singulex cTnI assay was determined by the mean +3 SD method. The normal range was determined on a population of 150 apparently healthy subjects. We also examined 56 serial samples from 17 patients who presented to the ED with a diagnosis of AMI in a preliminary study. Results were compared to the Centaur cTnI 1st (n=11) and 2nd (n=6) generation (ultra) assays. All had initial Centaur cTnI results that were <350 pg/mL (10%CV, 1st gen. Centaur), 6 were <100 pg/mL (99th percentile, 1st gen. Centaur), and 2 were <40 pg/mL (10% CV 2nd gen. Centaur). The cTnI concentration was positive on at least one subsequent serial samples from these patients on the Centaur, establishing the diagnosis of AMI.

Results: The analytical sensitivity of the Singulex assay was 0.2-0.3 pg/mL. The precision was <10% at 1.6, 12.5 and 50 pg/mL. The assay provided a linear response from 0.39 to >100 pg/mL (r2 = 0.99; y=1x+0.14). The reference population exhibited a normal distribution with a mean at 2 pg/mL (range 0.4-9 pg/mL). We established a preliminary cutpoint at 10 pg/mL, which is 50 fold higher than the analytical sensitivity. In the 9 cases that had initial Centaur cTnI value between 100 and 300 pg/mL, all were positive for Singulex with values ranging from 37-91 pg/ml (3.7-9 times >cut point). In the 8 cases that had initial Centaur cTnI value <100 pg/mL, 3 of 8 cases were Singulex positive. The Singulex assay was positive at least 1 sample earlier than the 10%CV cutoffs for either the 1st gen (10 of 11 patients) or 2nd gen Centaur (1 of 6 patients) assays.

Conclusion: We demonstrated a cTnI reference range between 0.4 and 9 pg/ml, with a mean of 2 pg/ml, some 10-fold higher than analytical sensitivity. Monitoring increases in cTnI levels above this reference range enabled detection of AMI several hours earlier than the Centaur cTnI assays.

Background: The European and American Cardiology Societies (ESC/ACC) have established cardiac troponin (cTnI) as the gold standard for diagnosis of myocardial infarction (AMI) and risk stratification for adverse cardiac events. The ESC/ACC recommends a cTnI cutoff at the 99th% of the normal range with an assay imprecision (CV) of <10%. Currently none of the commercial assays can detect cTnI in healthy subjects with the requisite precision, therefore many have advocated a cTnI cutoff at the 10% CV value. We developed and evaluated a high-sensitivity cTnI assay and determined its analytical and preliminary clinical performance.

Methods: The Singulex cTnI assay utilizes a 384-well ELISA plate, and the ZeptX™ Digital Molecule Counting (DMC) System. This assay uses a monoclonal capture antibody and a fluorescently-tagged affinity-purified goat detecting antibody. After washing, the fluorescently- tagged antibody is chemically released into each well. An aliquot is pumped into the analyzer. Individually-labeled antibodies are measured during capillary flow by setting the interrogation volume such that the emission of only 1 fluorescent molecule is detected in a defined space following laser excitation. With each signal representing a digital event, this configuration enables extremely high analytical sensitivities. Total fluorescent signal is determined as a sum of the individual digital events. Each molecule counted is a positive data point with hundreds to thousands of DMC events/sample. The limit of detection the Singulex cTnI assay was determined by the mean +3 SD method. The normal range was determined on a population of 88 apparently healthy subjects. This assay was correlated to the Bayer Centaur on 130 samples from patients admitted with chest pain. We also examined 47 serial samples from 18 patients who presented to the ED with a diagnosis of AMI. In this latter group, all had initial Centaur cTnI results that were <0.35 ng/mL (10% CV cutpoint), and 12 were <0.1 ng/mL (99th%). The cTnI concentration was positive on all subsequent serial samples from these patients on the Centaur, establishing the diagnosis of AMI.

Results: The analytical sensitivity of the Singulex assay was 1 pg/mL. The precision was 10% at 4 and 12 pg/mL. The reference population exhibited a normal distribution. The 99th percentile was determined to be <7 pg/mL. The linear regression between Singulex (y) and Centaur (x) was: y=0.113x + 0.048, r=0.937. In the 3 cases that had initial Centaur cTnI value between 0.1 and 0.35 ng/mL, all were positive for Singulex. In the 12 cases that had initial Centaur cTnI value <0.1 ng/mL, 5 of 12 cases were positive. The prospective use of the Singulex assay would have detected 53% more AMI cases than the Centaur when the admission sample was tested.

Conclusion: The use of a highly sensitive and precise cTnI assay will enable detection of AMI earlier than with existing cTnI assays. A higher number of patients at risk for adverse cardiac events may be detected. The increased sensitivity was achieved by counting individual fluorescent emission events by the ZeptX analyzer.