Single Molecule Counting Technology Research Articles

Highly sensitive single-molecule counting technology for cytokeratin 18 detection in non-alcoholic fatty liver disease using novel antibodies

Non-alcoholic fatty liver disease (NAFLD) affects approximately 25 % of the global population, presenting a significant threat to human health and imposing a substantial economic burden on society. Cytokeratin 18 (CK18) has emerged as a promising non-invasive biomarker for early detection of NAFLD. However, current diagnostic methods face challenges in accurately identifying NAFLD in its early stages. To address this issue, our study aimed to generate new CK18 antibodies and utilize a highly sensitive single-molecule immunodetection system customized for NAFLD. We embarked on a process involving the production of novel CK18 monoclonal antibodies, which were subsequently engineered into recombinant forms. In comparison to commercial antibodies, our antibodies exhibited superior affinity. Grounded in the SMCxPRO platform, our assay underwent rigorous assessment, adhering to well-defined biomarker assay criteria. The assay demonstrated specificity to CK18 and achieved a lower limit of quantification of 1 pg/ml. Importantly, elevated serum CK18 levels were observed among NAFLD patients compared to healthy individuals. The single-molecule detection system, utilizing our novel antibodies, holds significant promise for the early-stage diagnosis, dynamic disease monitoring, and prognostic evaluation of NAFLD patients.

A highly sensitive and quantitative assay for dystrophin protein using Single Molecule Count Technology

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle loss caused by mutations in dystrophin, resulting in decreased dystrophin levels. Dystrophin protein expression is a biomarker used to evaluate treatments that restore patient dystrophin levels. Currently, a semiquantitative assay using western blotting, which normalizes dystrophin expression to that of a control population, is used for regulatory filing. However, the current methods are limited in terms of sensitivity, quantification, and reproducibility. To address this, a highly sensitive and quantitative sandwich immune assay using Single Molecule Counting technology was established, with recombinant dystrophin protein as the calibrator. Capture and detection antibodies were selected to detect full-length dystrophin. Using this optimized assay, dystrophin levels in muscle samples from Myotonic Dystrophy (n = 9) and DMD (n = 8) subjects were 93.2 ± 31.9 (range: 49.4–145.3) and 14.5 ± 6.8 (range: 6.18–22.6) fmol/total protein mg, respectively. The lowest concentration of dystrophin measured in the DMD samples was 5 times higher than that in the lower limit of quantitation, a level not detected by western blotting. These data indicate that this assay accurately and sensitively measured dystrophin protein and may be useful in clinical trials assessing dystrophin restoration therapies.

Skin Interstitial Fluid and Plasma Multiplex Cytokine Analysis Reveals IFN-γ Signatures and Granzyme B as Useful Biomarker for Activity, Severity and Prognosis Assessment in Vitiligo.

BackgroundThe course of vitiligo is unpredictable, with periods of disease flare-ups and prolonged recovery periods. It is essential to establish a biomarker profile as a substitute marker for disease activity to predict disease activity, severity, and prognosis prediction. The use of localized skin interstitial fluid as biomarkers has recently gained interest, but extensive studies of the association between skin interstitial fluid, plasma, and the disease course is lacking. This study aims to evaluate the cytokine expression profiles in the skin and plasma and the utility of the biomarker panel in assessing disease activity, severity, and prognosis in patients with vitiligo.MethodsIn this prospective cohort study, 86 patients and 34 healthy controls were recruited from the outpatient department of a tertiary medical center from March 2019 to September 2021. All patients were of Asian ethnicity. Two independent investigators evaluated disease activity and severity with longitudinal follow-ups for treatment response for a-12 month period. Ultrasensitive multiplex cytokine panel and single-molecule counting technology immunoassays were used to study the cytokine expression in skin interstitial fluid and plasma.ResultsIFN-γ and its’ signature cytokines, including CXCL9, CXCL10, and GzmB, are most highly expressed in the vitiligo patients’ lesion skin interstitial fluid and plasma compared to healthy control. By way of comparison, no significant changes in IL-1β, IL-13, IL-15, IL-17A, IL-18 were observed. Receiver operating characteristic analysis revealed that IFN-γ is the most sensitive and specific marker in predicting disease activity, followed by CXCL10 and GzmB. CXCL-9 was sensitive and specific in diagnosing vitiligo disease severity. The decrease in IFN-γ expression level is positively correlated with the treatment response.ConclusionIFN-γ, CXCL9, CXCL10, and GzmB are highly expressed in vitiligo patients’ lesion skin and plasma and may serve as biomarkers for the clinical activity, severity, and prognosis prediction in vitiligo patients. Among all, IFN-γ exerts the highest predictive value in disease activity and treatment response, supporting the critical role of IFN-γ in the pathogenesis of vitiligo.

MosaicNeedles: A tool for multi-omic liquid biopsy sensitivity and specificity enhancement.

e15019 Background: Liquid biopsy has evolved to be an important method complementary to tissue biopsy. It is not only non-invasive, but also has the potential to detect cancer in its earliest stages and monitor patients in remission. The integration of proteomics into liquid biopsy may transform the molecular diagnostics of cancer and accelerate basic and clinical oncology research. A recent study showed that adding just 8 protein biomarkers to a panel of circulating DNA biomarkers increased the diagnostic accuracy up to 98% sensitivity and 99% specificity. Proteomics also bridges the gaps of functional information lost due to post-transcriptional and post-translational modifications in the genomic approach. However, the proteogenomic approach normally requires the use of multiple different assay technologies and laboratory workflows, including mass spectrometry. Methods: NanoMosaic’s Tessie platform employs a densely integrated nanoneedle sensor array (thus named MosaicNeedles) which can be used to detect both nucleic acids and proteins in a single assay process with reduced workflow complexity, without the need for mass spectrometry. Results: The NanoMosaic platform is a label-free, digital, single molecule counting technology using nanoneedles. It achieves sub-pg/ml (̃fM) level sensitivity with 7 logs of dynamic range. An array of nanoneedles is densely integrated and manufactured with CMOS-compatible nanofabrication processes. Each nanoneedle is a single molecule biosensor that is functionalized with capture probes. The capture probe can be either an antibody for protein detection or an oligonucleotide with a specific target sequence to a DNA fragment, mRNA, or miRNA of interest. The scattering spectrum of each nanoneedle changes when an analyte binds to its surface. At low abundance, analytes that are captured can be quantitated by counting the presence or absence of a color change on each individual nanoneedle in a binary fashion. As an analyte concentration increases the binding events increase accordingly and achieve saturation. In this range, an analog analysis on the spectrum shift will be performed, thus providing a wider dynamic range, up to 7 logs. Ultrahigh level multiplex can be achieved by parallelizing each analyte specific sensing area without loss of sensitivity or dynamic range. A 10,000-plex study can be achieved with a total of 2.5 billion nanoneedles on a 50mm by 50mm consumable. In this consumable, a 2,000-plex proteome and 8,000 cell-free DNA fragments can be detected. Conclusions: In conclusion, a full proteogenomic quantification can be performed on the NanoMosaic platform in one reaction, with higher sensitivity, lower cost and higher throughput than is currently possible by traditional methods. In addition, the high-plexibility of the NanoMosaic platform allows the discovery of new biomarkers across the whole proteome without the need for mass spectrometry.

Phospho-S129 Alpha-Synuclein Is Present in Human Plasma but Not in Cerebrospinal Fluid as Determined by an Ultrasensitive Immunoassay.

Accumulation and aggregation of misfolded alpha-synuclein is believed to be a cause of Parkinson’s disease (PD). Phosphorylation of alpha-synuclein at S129 is known to be associated with the pathological misfolding process, but efforts to investigate the relevance of this post-translational modification for pathology have been frustrated by difficulties in detecting and quantifying it in relevant samples. We report novel, ultrasensitive immunoassays based on single-molecule counting technology, useful for detecting alpha-synuclein and its S129 phosphorylated form in clinical samples in the low pg/ml range. Using human CSF and plasma samples, we find levels of alpha-synuclein comparable to those previously reported. However, while alpha-synuclein phosphorylated on S129 could easily be detected in human plasma, where its detection is extremely sensitive to protein phosphatases, its levels in CSF were undetectable, with a possible influence of a matrix effect. In plasma samples from a small test cohort comprising 5 PD individuals and five age-matched control individuals we find that pS129 alpha-synuclein levels are increased in PD plasma samples, in line with previous reports. We conclude that pS129 alpha-synuclein is not detectable in CSF and recommend the addition of phosphatase inhibitors to plasma samples at the time of collection. Moreover, the findings obtained on the small cohort of clinical plasma samples point to plasma pS129 alpha-synuclein levels as a candidate diagnostic biomarker in PD.

Establishing the 99th percentile for high sensitivity cardiac troponin I in healthy blood donors from Southern Italy.

IntroductionThe knowledge of high sensitivity cardiac troponin I (hsTnI) distribution in a reference population is mandatory for its introduction in clinical practice. The aim of this study was to define the Upper Reference Limit (URL) of hsTnI measured by Single Molecule Counting technology (SMC) in an accurately selected reference population.Materials and methodsIn the study 1140 blood donors were included and selected on the basis of medical history and biomarkers. High sensitivity cardiac troponin I was measured by SMC technology (Clarity, Singulex, Alamed, USA). The 99th percentile was calculated by the non-parametric method according to the Clinical and Laboratory Standard Institute - CLSI C28-A3.ResultsThe median age was 41 years (IQR: 28 - 50) and 69% were males. The overall 99th percentile was 5 ng/L (90% CI: 4.2 - 5.6). When considering sex-related differences, we found slight differences between the 99th percentile in males and females. Moreover, the 99th percentile trended with age, especially in females.ConclusionsWe defined the 99th percentile of hs-cTnI measured by SMC technology in a highly selected healthy population, with only minor differences between males and females. Our findings provide the basic criteria for the reliable interpretation of hsTnI concentrations measured by the SMC technology in clinical settings.

Laboratory comparison between cell cytotoxicity neutralization assay and ultrasensitive single molecule counting technology for detection of Clostridioides difficile toxins A and B, PCR, enzyme immunoassays, and multistep algorithms

Diagnostic tests for Clostridioides difficile infection (CDI) lack either specificity (nucleic acid amplification tests) or sensitivity (enzyme immunoassays; EIAs). The performance of the Singulex Clarity® C. diff toxins A/B assay was compared to cell cytotoxicity neutralization assay. Testing was also performed using an EIA for glutamate dehydrogenase (GDH) and C. difficile toxins A and B (C. Diff Quik Chek Complete®), polymerase chain reaction (PCR) (BD MAX™ Cdiff Assay), and 2 multistep algorithms: algorithm 1 (discordant GDH/toxin results arbitrated by PCR) and algorithm 2 (PCR-positive samples tested with toxin EIA). The Clarity assay and PCR both had 97% sensitivity, while specificity was 100% for Clarity and 79% for PCR. Algorithm 1 yielded 41% discordant results, and both toxin EIA and algorithm 2 had 58% sensitivity. Median toxin concentrations, as measured by the Clarity C. difficile toxin assay, were 3590, 11.5, 0.4, and 0 pg/mL for GDH+/toxin+, GDH+/toxin−/PCR+, GDH+/toxin−/PCR−, and GDH−/toxin− samples, respectively (P < 0.001). The Clarity assay may offer a single-test solution for CDI.

Cardiovascular stress biomarker assessment of middle-aged non-athlete marathon runners.

Cardiovascular safety of marathon running in recreational runners remains unclear. We set up this study to comprehensively evaluate the effects of a marathon run on the profile of cardiovascular stress biomarkers in connection with transthoracic echocardiography. Thirty-three healthy male amateur runners, aged ≥50 years (mean age 57 ± 7) were enrolled. Venous blood samples were obtained before the marathon, just after the race, 2-4 and seven days after the marathon. Using novel single molecule counting technology we measured: plasma concentrations of high-sensitivity cardiac troponin I (hs-cTnI) and endothelin-1. N-terminal pro B-type natriuretic peptide was measured using electrochemiluminescence. Each participant had transthoracic echocardiography before and immediately after the race. We observed a sharp rise in the levels of all biomarkers after the race (all p < 0.01), which subsequently normalized after 2-4 days and stayed within normal range after seven days. Runners with intensive training programmes (>169 km/month, a median for the studied group) had lower hs-cTnI leak after the race (median 15.11 ng/ml and 31.2 ng/ml, respectively; p < 0.05). Neither transthoracic echocardiography measures of ventricles nor strain rates changed after the run. We observed a decrease in the left atrial volume index and increase in the maximal right atrial volumes (all p < 0.05). Changes in Doppler indices of diastolic function suggestive of alteration in left ventricular relaxation were observed. Marathon run is associated with sharp and significant rises in the biomarkers of cardiovascular stress. The profile of these changes, however, along with echocardiographic parameters, does not suggest irreversible myocardial damage.

Highly sensitive multiplex protein detection by droplet‐free digital ELISA

AbstractDigital enzyme‐linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. On the other hand, we have developed a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In this study, we present a method that enables the multiplex detection of proteins based on Droplet‐free Digital ELISA. First, two types of paramagnetic beads immobilized with antibodies specific to different target proteins were incubated with a sample solution, and target proteins were captured on their specific beads. Then, these beads were labeled with horseradish peroxidase (HRP), and tyramide substrate reacted with HRP on beads, resulting in products of this reaction deposited on those beads. This signal amplification on beads makes it possible to count the number of labeled beads digitally. We used this approach to simultaneously detect IL‐6 and HBs Ag with single molecule resolution. The obtained limit of detections were 0.1 pg/mL and 0.013 IU/mL, respectively. Our method has potential applications in simple in vitro diagnostic systems for simultaneous and high‐sensitive detection of protein biomarkers.

Clinical utility of single molecule counting technology for quantification of KIM-1 in patients with heart failure and chronic kidney disease

BackgroundAcute kidney injury (AKI) is associated with high morbidity and mortality, and may lead to chronic kidney disease (CKD). Traditional serum biomarkers for acute and chronic renal dysfunction are insensitive and nonspecific. While urinary kidney injury molecule-1 (KIM-1) is a sensitive and specific measure of kidney tubular injury, it is difficult to obtain in acute settings. Thus, our objective was to develop a highly sensitive immunoassay for plasma KIM-1. MethodsA novel plasma KIM-1 immunoassay was developed using Single Molecule Counting technology (SMC). It was clinically validated in: 120 healthy subjects to establish a preliminary reference range; 25 healthy subjects to assess biological variability; 200 patients with heart failure (CHF); and 60 patients from a CKD case control. ResultsSMC KIM-1 assay provided a limit of detection of 1.4pg/mL (reporting range from 2pg/mL to 1000pg/mL). Inter-assay precision was 9–15% CV. Median KIM-1 value in healthy subjects was 119pg/mL with a RR 95th percentile of 292pg/mL. KIM-1 demonstrated low weekly biological variability over 6weeks. KIM-1 was elevated in patients with CKD or CHF. Adjusted odds ratios for differentiating CHF or CKD from controls were 9.6 (95% CI 2.7–35.0) and 3.6 (95% CI 1.1–11.6), respectively. In CHF, KIM-1 values correlated inversely with eGFR (Spearman R=−0.32, p<0.0001). ConclusionsPlasma KIM-1 is quantifiable in healthy volunteers, elevated in CKD and CHF patients, and correlates with eGFR. Additional investigation is needed to determine if KIM-1 provides prognostic value for CKD and CHF patient outcomes.

Subclinical sleep apnoea and plasma levels of endothelin-1 among young and healthy adults

ObjectiveObstructive sleep apnoea (OSA) is a risk factor for vascular disease and other adverse outcomes. These associations may be at least partly due to early endothelin-1 (ET-1)-mediated endothelial dysfunction (ED)....

Urinary levels of novel kidney biomarkers and risk of true worsening renal function and mortality in patients with acute heart failure.

Recent studies indicate the need to redefine worsening renal function (WRF) in acute heart failure (AHF), linking a rise in creatinine with clinical status to identify patients who develop 'true WRF'. We evaluated the usefulness of serial assessment of urinary levels of neutrophil gelatinase-associated lipocalin (uNGAL), kidney injury molecule-1 (uKIM-1), and cystatin C (uCysC) for prediction of 'true WRF'. In 132 patients with AHF, uNGAL, uKIM-1, and uCysC were measured using a highly sensitive immunoassay based on a single-molecule counting technology (Singulex, Alameda, CA, USA) at baseline, day 2, and day 3. Patients who developed WRF (a ≥0.3 mg/dL increase in serum creatinine or a >25% decrease in the estimated glomerular filtration rate from the baseline value) were differentiated into those 'true WRF' (presence of deterioration/no improvement in clinical status during hospitalization) vs. 'pseudo-WRF' (uneventful clinical course). 'True WRF' occurred in 13 (10%), 'pseudo-WRF' in 15 (11%), whereas the remaining 104 (79%) patients did not develop WRF. Patients with 'true WRF' were more often females, had higher levels of NT-proBNP, creatinine, and urea on admission, higher urine albumin to creatinine ratio at day 2, higher uNGAL at baseline, day 2, and day 3, and higher KIM-1 at day 2 (vs. pseudo-WRF vs. without WRF, all P < 0.05). Patients with pseudo-WRF did not differ from those without WRF. In the multivariable model, elevated uNGAL at all time points and uKIM-1 at day 2 remained independent predictors of 'true WRF'. Elevated levels of uNGAL and uKIM-1 may predict development of 'true WRF' in AHF.

Elevated troponin I level assessed by a new high-sensitive assay and the risk of poor outcomes in patients with acute heart failure

BackgroundThe interpretation and clinical usefulness of elevated levels of cardiac troponins in acute heart failure (AHF) remain controversial. We aimed to characterize the relationship between changes in cardiac troponin I (measured using a new high-sensitive immunoassay by single-molecule counting technology, Singulex, Alameda, USA; hs-TnI) during first 48h of hospital stay and patients' characteristics and the outcomes. Methods and resultsWe measured hs-TnI at baseline, after 24 and 48h in 130 AHF patients (mean age: 65±13years, 77% men). The percentage of patients with elevated hs-TnI (i.e., above the upper reference limit [URL]>10.19pg/mL) were: on admission – 59%, after 24h – 61%, and after 48h – 58%. Elevated baseline level of hs-TnI was associated with more severe dyspnoea on admission but neither peak level nor changes in hs-TnI during first 48h were related to the dyspnoea severity or magnitude of dyspnoea relief. During 1-year follow-up there were 32 (25%) cardiovascular deaths. Neither absolute baseline nor peak values of hs-TnI predicted cardiovascular mortality. Only changes in hs-TnI were independently associated with cardiovascular mortality with the strongest relationship seen in peak change in hs-TnI: patients with an increase vs. remaining patients - hazard ratio (95% confidence interval): 3.22 (1.52–6.82)p=0.002. ConclusionsUsing the new assay (proved to be more sensitive that the other available troponin assays) we observed that approximately 60% of patients with AHF presented elevated hs-TnI above URL during first 48h of hospital stay. Only significant increase in hs-TnI predicted cardiovascular mortality.

Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System.

Digital enzyme-linked immunosorbent assay (ELISA) is a single molecule counting technology and is one of the most sensitive immunoassay methods. The key aspect of this technology is to concentrate enzyme reaction products from a single target molecule in femtoliter droplets. This study presents a novel Digital ELISA that does not require droplets; instead, enzyme reaction products are concentrated using a tyramide signal amplification system. In our method, tyramide substrate reacts with horseradish peroxidase (HRP) labeled with an immunocomplex on beads, and the substrate is converted into short-lived radical intermediates. By adjusting the bead concentration in the HRP-tyramide reaction and conducting the reaction using freely moving beads, tyramide radicals are deposited only on beads labeled with HRP and there is no diffusion to other beads. Consequently, the fluorescence signal is localized on a portion of the beads, making it possible to count the number of labeled beads digitally. The performance of our method was demonstrated by detecting hepatitis B surface antigen with a limit of detection of 0.09 mIU/mL (139 aM) and a dynamic range of over 4 orders of magnitude. The obtained limit of detection represents a >20-fold higher sensitivity than conventional ELISA. Our method has potential applications in simple in vitro diagnostic systems for detecting ultralow concentrations of protein biomarkers.

Multiplex single molecule counting technology used to generate interleukin 4, interleukin 6, and interleukin 10 reference limits

Detecting biomarkers at pg/ml concentrations or below is, in many situations, critical for quantifying levels in healthy individuals as well as the changes that can occur in the progression of disease states. The ability to detect multiple biomarkers from the same sample allows for better diagnoses, more efficient testing, and lower volumes of sample required. Based on single molecule counting technology, a multiplex instrument was designed and built that is capable of detecting cytokines and other low-abundance proteins at sub-pg/ml quantities in human plasma samples. The multiplex single molecule counting instrument was used to generate 95% reference limits for interleukin 4 (IL-4, <0.61 pg/ml), interleukin 6 (IL-6, <6.53 pg/ml), and interleukin 10 (IL-10, <1.08 pg/ml) from 100 healthy human donor plasma samples, with more than 90% of IL-4 concentrations and 100% of IL-6 and IL-10 concentrations above the limit of detection.

Abstract 15934: Higher Midlife Serum Levels of Kidney-injury Molecule-1 (KIM-1) are Associated With Incident Fatal CHD Over Very Long-term Follow-up Among Men

Introduction: Higher levels of kidney-injury molecule-1 (KIM-1) measured in urine are associated with presence and progression of acute renal disease. A recent study reported similar results for KIM-1 measured in blood. Hypothesis: We hypothesized that KIM-1 measured in stored serum from middle-aged men who participated in the Multiple Risk Factor Intervention Trial (MRFIT) would differentiate very long-term risk of fatal CHD vs. survival to a mean age of 80 over approximately 30 year follow-up. Methods: We conducted a nested case-control study within MRFIT, which in 1973-76 randomized 12,866 high risk but CVD free men ages 35-57 to risk factor intervention vs. usual care. Serum samples were collected at baseline and stored for future use. The trial concluded in 1982 but long-term mortality follow-up was ascertained through 2005 using the National Death Index. From MRFIT participants with stored serum from baseline, we sampled 100 men who died of CHD (mean age 47.3 at baseline and 73.9 at death), and 100 men who survived to 2005 (mean age =48.4 at baseline and 80.1 in 2005.) KIM-1 was assayed from stored serum samples using high sensitivity single-molecule counting technology (Erenna ® Immunoassay System, Singulex), with limit of detection (LoD)=0.5 pg/ml, and lower limit of quantification (LLoQ)=2.0 pg/ml. Results were compared between cases and controls using Wilcoxon rank tests and logistic regression. Results: Inter-assay %CVs were 8%. Median KIM-1 was higher for smokers vs. non-smokers and for men with vs. without hypertension, but was not associated with high cholesterol. KIM-1 was significantly higher in cases (183 pg/ml (IQR: 137-239) versus controls, (161 pg/ml (IQR:109-212), p=0.03; OR (95%CI)for Q4 versus Q1 was 2.26 (1.02 - 5.02) Adjusted for age and smoking the OR(95%CI) of fatal CHD for Q4 vs. Q1 was 2.34 (1.02- 5.37), and further adjusted for diastolic BP and serum cholesterol at baseline, was 2.0 (95% CI: 0.8-4.7). Conclusions: Higher serum KIM-1 levels at midlife were associated with a ∼2-fold increased risk of fatal CHD vs. survival over ∼30 years of follow-up. This is the first report of a longitudinal association of circulating KIM-1 levels with fatal CHD in long-term follow-up.

Longitudinal studies of cardiac troponin I concentrations in serum from male cynomolgus monkeys: resting values and effects of oral and intravenous dosing on biologic variability.

There is a paucity of information regarding cardiac troponin (cTn) concentrations in peripheral blood of nonhuman primates (NHP). Even less is known regarding cTn concentrations in monkeys that are restrained for oral or intravenous (iv) dosing. The objectives of these studies were to (1) determine cardiac troponin I (cTnI) concentration in resting Cynomolgus monkeys and investigate biologic variability in cTnI concentration over time, (2) determine cTnI changes in restrained monkeys given sham oral dosing, and (3) determine cTnI changes in restrained NHP given a sham intravenous dosing. The Research Use Only Erenna cTnI ultrasensitive immunoassay based on single molecule counting technology was used to determine serum cTnI concentration in longitudinal studies of male Cynomolgus monkeys at rest, and after sham oral and intravenous dosing. Animals were catheterized prestudy, and blood samples were collected by an automated sampling device to limit disturbance of the animals during studies. In resting monkeys cTnI concentrations were relatively low and constant and ranged from 0.2 to 9.6 pg/mL (mean = 2.5 pg/mL), with minimal variability during a 24-hour period. Animals given sham oral dosing also had low cTnI concentration with little variability similar to the resting values. Several animals restrained for intravenous dosing had a small transient increase in cTnI concentration (~5-25 pg/mL) that resolved quickly within one to 3 hours postinjection. Results of this longitudinal study provide information that may be important in differentiating effects of animal handling from those associated with compound-related effects in preclinical toxicology studies of drugs in development.

Plasma endothelin-1 and cardiovascular risk among young and healthy adults

ObjectivesEndothelial dysfunction is a major precursor of atherosclerosis. The aim of this study was to assess the interrelationships between plasma endothelin-1 (ET-1) levels and cardiovascular risk among young and healthy individuals. MethodsWe performed a population-based study among 2160 healthy adults aged between 25 and 41 years in the Principality of Liechtenstein. Individuals with prevalent cardiovascular disease, diabetes or a body mass index >35 kg/m2 were excluded. Plasma ET-1 was measured using a novel high-sensitive, single-molecule counting technology. The relationships between plasma levels of ET-1 and various cardiovascular risk factors were assessed by multivariable regression analyses. ResultsMedian age of our population was 37 years. Median ET-1 levels across ET-1 quartiles were 1.86, 2.33, 2.76 and 3.48 pg/mL. After multivariable adjustment, there were significant correlations of ET-1 with systolic blood pressure (β per 1-unit increase in log transformed ET-1 2.30 (95% confidence interval (CI) 1.03; 3.58, p = 0.0004), C-reactive protein (β 0.19 (95% CI 0.03; 0.34, p = 0.021), glomerular filtration rate (β −1.73 (95% CI −3.17; −0.29, p = 0.019), and current smoking (Odds ratio 1.94 (95% CI 1.39; 2.71, p < 0.0001). We also found a highly significant association between ET-1 levels and overall cardiovascular risk estimated by the “Prospective Cardiovascular Münster” (PROCAM) and the Framingham score (β 0.18 (95% CI 0.06; 0.31, p = 0.004, and β 0.11 (95% CI 0.05; 0.16), p < 0.0001, respectively). ConclusionsPlasma ET-1 levels are easily measurable in healthy adults and correlate with major cardiovascular risk factors and global cardiovascular risk.

QUANTIFYING MUTANT HUNTINGTIN IN HUNTINGTON'S DISEASE CSF

Huntington9s disease (HD) is a fatal, autosomal dominant neurodegenerative disease. No disease-modifying treatments exist, but oligonucleotide-based ‘gene silencing’ approaches that aim to reduce expression of the causative mutant huntingtin (mHTT) protein are in advanced development. Low-abundance mHTT has never been quantified in the patient CNS, limiting our understanding of its role in the neuropathobiology of HD in vivo, and precluding the demonstration of target engagement by HTT-lowering drugs. We developed a novel ultra-sensitive mHTT immunoassay using single-molecule counting technology. Using cerebrospinal fluid (CSF) collected from 9 HD mutation carriers and 3 controls, under strictly standardised conditions, mHTT was successfully quantified and its levels completely distinguished controls, premanifest mutation carriers and manifest HD patients. mHTT level was significantly associated with disease burden score, a measure of lifetime exposure to mHTT, suggesting that soluble mHTT species increase in concentration with disease progression. An independent association with CSF neurofilament light chain level suggests the mHTT detected is neuronal in origin. Quantifying mHTT in CSF may provide a means of monitoring disease progression in HD. Our novel assay will be refined using larger CSF collections, and used to better understand the neuropathobiology of HD and to support clinical trials of disease-modifying HD therapeutics.

Short- and Long-Term Cardiac Troponin I Analyte Stability in Plasma and Serum from Healthy Volunteers by Use of an Ultrasensitive, Single-Molecule Counting Assay

Cardiac troponin I (cTnI)1 is the gold standard biomarker for diagnosing patients with acute coronary syndromes. Expert panels have defined the decision limit at >99th percentile of a reference population and assay imprecision of ≤10%. Prototype ultrasensitive cTnI assays allow accurate detection of low concentrations of cTnI in healthy individuals. However, these novel assays require analytical and biological validation before they can be put into clinical practice. We previously reported the 99th percentile cutoff of 7 ng/L for the Erenna® cTnI immunoassay (Singulex), with the requisite precision. Using a single-molecule counting technology, the limit of detection (0.2 ng/L) surpassed many other commercial assays. The improved limit of detection is not an artifact of nonspecific-binding events (1). We also showed that the increased analytical sensitivity of the assay enabled measurement of the short- and long-term biological variation of cTnI from healthy individuals (2). However, to what extent minute levels of analyte instability may have contributed to this variation was not established. We determined the in vitro stability of cTnI in whole blood and serum over short- and long-term intervals in both healthy subjects and in a small subset of patients who presented to the San Francisco General Hospital Emergency Department. A protocol for the use of leftover blood from routine collections and for collecting blood of healthy …