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

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

Recommendations for the use of cardiac troponin (cTn) measurement in acute cardiac care have recently been published.1 Subsequently, a high-sensitivity (hs) cTn T assay was introduced into routine clinical practice.2 This assay, as others, called highly sensitive, permits measurement of cTn concentrations in significant numbers of apparently illness-free individuals. These assays can measure cTn in the single digit range of nanograms per litre (=picograms per millilitre) and some research assays even allow detection of concentrations 10% at the 99th percentile URL limiting that ability.5–7 However, the less precise cTn assays do not cause clinically relevant false-positive diagnosis of acute myocardial infarction (AMI) and a CV <20% at the 99th percentile URL is still considered acceptable.8

We believe that hs-cTn assays, if used appropriately, will improve clinical care. We propose criteria for the clinical interpretation of test results based on the limited evidence available at this time.

‘Sensitive’ and ‘high-sensitive’ are terms often used by manufacturers to describe their assays for marketing purposes. In some cases, it reflects higher sensitivity than former assays developed by the same company, and in other situations it reflects a higher sensitivity than most assays on the market. Although there is still no consensus regarding when the terms ‘sensitive’ and ‘high-sensitive’ should be applied, we advocate that cTn assays should be labelled ‘high-sensitive’ only if they fulfil the analytical criteria suggested by …

How to use high-sensitivity cardiac troponins in acute cardiac care

Background—Biomarkers for predicting cardiovascular events in community-based populations have not consistently added information to standard risk factors. A limitation of many previously studied biomarkers is their lack of cardiovascular specificity. Methods and Results—To determine the prognostic value of 3 novel biomarkers induced by cardiovascular stress, we measured soluble ST2, growth differentiation factor-15, and high-sensitivity troponin I in 3428 participants (mean age, 59 years; 53% women) in the Framingham Heart Study. We performed multivariable-adjusted proportional hazards models to assess the individual and combined ability of the biomarkers to predict adverse outcomes. We also constructed a “multimarker” score composed of the 3 biomarkers in addition to B-type natriuretic peptide and high-sensitivity C-reactive protein. During a mean follow-up of 11.3 years, there were 488 deaths, 336 major cardiovascular events, 162 heart failure events, and 142 coronary events. In multivariable-adjusted ...

Prognostic Utility of Novel Biomarkers of Cardiovascular Stress: The Framingham Heart Study

Background: Endurance exercise can increase cardiac troponin (cTn) concentrations as high as those seen in cases of minor myocardial infarction. The inability of most cTn assays to reliably quantify cTn at very low concentrations complicates a thorough data analysis, and the clinical implications of such increases remain unclear. The application of recently developed highly sensitive cTn immunoassays may help resolve these problems.

Methods: We evaluated the precommercial highly sensitive cardiac troponin T (hs-cTnT) assay from Roche Diagnostics and the Architect cardiac troponin I (cTnI-Architect) assay from Abbott Diagnostics by testing samples from a reference population of 546 individuals and a cohort of 85 marathon runners. We also measured the samples with the current commercial cTnT assay for comparison.

Results: Although the hs-cTnT and cTnI-Architect assays were capable of measuring cTn concentrations at low concentrations (<0.01 μg/L), only the hs-cTnT assay demonstrated a CV of <10% at the 99th percentile of the reference population and a near-gaussian distribution of the measurements. After a marathon, 86% of the runners had cTnT concentrations greater than the 99th percentile with the hs-cTnT assay, whereas only 45% of the runners showed increased concentrations with the current cTnT assay. cTn concentrations remained significantly increased the day after the marathon. A multiple regression analysis demonstrated marathon experience and age to be significant predictors of postmarathon cTn concentrations ( P < 0.05).

Conclusions: The hs-cTnT assay was the only assay tested with a performance capability sufficient to detect cTn concentrations in healthy individuals. The number of runners with increased cTn concentrations after a marathon depends highly on an assay’s limit of detection (LOD). The assay with the lowest LOD, the hs-cTnT assay, showed that almost all runners had increased cTn concentrations. The clinical implications of these findings require further investigation.

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Reference population and marathon runner sera assessed by highly sensitive cardiac troponin T and commercial cardiac troponin T and I assays.

Purpose Doxorubicin causes cardiac injury and cardiomyopathy in children with acute lymphoblastic leukemia (ALL). Measuring biomarkers during therapy might help individualize treatment by immediately identifying cardiac injury and cardiomyopathy. Patients and Methods Children with high-risk ALL were randomly assigned to receive doxorubicin alone (n = 100; 75 analyzed) or doxorubicin with dexrazoxane (n = 105; 81 analyzed). Echocardiograms and serial serum measurements of cardiac troponin T (cTnT; cardiac injury biomarker), N-terminal pro-brain natriuretic peptide (NT-proBNP; cardiomyopathy biomarker), and high-sensitivity C-reactive protein (hsCRP; inflammatory biomarker) were obtained before, during, and after treatment. Results cTnT levels were increased in 12% of children in the doxorubicin group and in 13% of the doxorubicin-dexrazoxane group before treatment but in 47% and 13%, respectively, after treatment (P = .005). NT-proBNP levels were increased in 89% of children in the doxorubicin group and in...

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Changes in Cardiac Biomarkers During Doxorubicin Treatment of Pediatric Patients With High-Risk Acute Lymphoblastic Leukemia: Associations With Long-Term Echocardiographic Outcomes

Background: Immunoassay (IA) technology has expanded the clinical utility of protein biomarkers, but demands for increased sensitivity, dynamic reporting ranges, and small sample volumes have limited the potential clinical usefulness of many biomarkers. We assessed the performance, including limits of detection (LODs) and the dynamic reporting range, of an IA-based technology, Erenna Immunoassay System, for a series of biomarkers, including cardiac troponin I (cTnI). Methods: Erenna IAs were used with 10 different and clinically important biomarkers to ascertain the LOD with various sample sizes (10 μL to 200 μL). Results: The Erenna Immunoassay System generated LODs of 10–100 pg/L using 100 μL of sample. For cTnI, the LOD was 0.2 ng/L and a 10% CV was seen between 0.78 and 1.6 ng/L. Conclusions: The Erenna IA-based technology reproducibly measures protein biomarkers with detection limits of 10–100 pg/L, with a dynamic range of >4.5 logs in sample volumes of 50–150 μL.

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Ultrasensitive Flow-based Immunoassays Using Single-Molecule Counting

The invention encompasses analyzers and analyzer systems that include a single particle analyzer, methods of using the analyzers and analyzers systems to analyze samples, either for single particles or for multiple particles (multiplexing), methods of doing business based on the use of the analyzers or analyzer systems of the system, and electronic media for storing parameters useful in the analyzers and analyzer systems of the invention.

System and methods for sample analysis

Disclosed are methods, kits, and compositions for the highly sensitive detection of molecules. The methods, kits, and compositions are useful in determining concentrations of molecules in samples to levels of 1 femtomolar, 1 attomolar, or lower. The methods, kits, and compositions also allow the determination of concentration over a wide range, e.g., 7-log range, without need for sample dilution.

Methods and Compositions for Highly Sensitive Detection of Molecules

Cardiovascular disease, e.g., congestive heart failure, is often first diagnosed after the onset of clinical symptoms, eliminating potential for early intervention. The invention provides a multi-marker immunoassay, including cardiac pathology and vascular inflammation biomarkers, yielding a more sensitive assay for early detection of CHF in plasma. A panel consisting of cardiac pathology (cTnI, BNP) and vascular inflammation (IL-6, TNFα, IL-17a) biomarkers provided a sensitivity of 94% for association with CHF.

Highly sensitive biomarker panels

Disclosed are methods, kits, and compositions for fluorescent detection of single biomolecules, e.g. proteins, using highly sensitive fluorescent dyes and a flow cell with one interrogation space for irradiation by focused laser beam. The methods, kits, and compositions are useful in determining concentrations of molecules in samples to levels of 1 femtomolar, 1 attomolar, or lower. The methods, kits, and compositions also allow the determination of concentration over a wide range, e.g., 7-log range, without need for sample dilution.

Richard Livingston

Papers

Ultrasensitive Flow-based Immunoassays Using Single-Molecule Counting

System and methods for sample analysis

Methods and Compositions for Highly Sensitive Detection of Molecules

Highly sensitive biomarker panels

Methods and compositions for highly sensitive analysis of markers and detection of molecules