High-sensitivity detection of cardiac troponin I with UV LED excitation for use in point-of-care immunoassay.

High-sensitivity cardiac troponin assay development enables determination of biological variation in healthy populations, more accurate interpretation of clinical results and points towards earlier diagnosis and rule-out of acute myocardial infarction. In this paper, we report on preliminary tests of an immunoassay analyzer employing an optimized LED excitation to measure on a standard troponin I and a novel research high-sensitivity troponin I assay. The limit of detection is improved by factor of 5 for standard troponin I and by factor of 3 for a research high-sensitivity troponin I assay, compared to the flash lamp excitation. The obtained limit of detection was 0.22 ng/L measured on plasma with the research high-sensitivity troponin I assay and 1.9 ng/L measured on tris-saline-azide buffer containing bovine serum albumin with the standard troponin I assay. We discuss the optimization of time-resolved detection of lanthanide fluorescence based on the time constants of the system and analyze the background and noise sources in a heterogeneous fluoroimmunoassay. We determine the limiting factors and their impact on the measurement performance. The suggested model can be generally applied to fluoroimmunoassays employing the dry-cup concept.

Autoantibodies to cardiac troponin associate with higher initial concentrations and longer release of troponin I in acute coronary syndrome patients.

BACKGROUND: Cardiac troponin (cTn) is an established marker of myocardial infarction. Pronounced heterogeneity and the minute amounts released into the circulation constitute significant challenges for cTn detection. Recently, autoantibody formation to cTn was shown to be common and to interfere with immunoassay performance. In this study, we investigated cTn autoantibodies and cardiac troponin I (cTnI) in acute coronary syndrome (ACS) patients over a 1-year period after the index event. METHODS: We used a second-generation cTnI assay designed to reduce the interference of cTn autoantibodies. The assay for cTn autoantibodies used 2 anti-cTnI antibodies to capture the ternary cTnI-complex, enabling unrestricted binding of the autoantibodies, which were detected with a labeled antihuman IgG antibody. We analyzed serum samples from 81 non-ST-elevation ACS patients taken at admission and after 1 week and 3 and 12 months. RESULTS: We found 14 cTn autoantibody-positive patients (21%) among the 67 cTnI-positive and none among the 14 cTnI-negative patients. Nine were autoantibody-positive at admission, and 5 became positive at 1 week. Autoantibody signals significantly increased in the 1-week and 3-month samples. At all time points, cTnI was significantly increased in the autoantibody-positive group relative to the negative group. Persistent cTnI elevations at 3 and 12 months were seen in the patients already autoantibody positive at admission. CONCLUSIONS: During ACS, patients with cTn autoantibodies have higher cTnI release and therefore larger myocardial damage than patients without autoantibodies. Their cTnI release also lasts longer, at least months. The possible prognostic impact of these observations must be evaluated in larger clinical cohorts.

Early markers of myocardial injury: cTnI is enough.

BACKGROUND: We compared the early diagnostic and prognostic performance of a highly sensitive cardiac troponin I (cTnI) assay with heart-type fatty acid binding protein (H-FABP), in the early hours of acute coronary syndrome. METHODS: Serum samples of 293 patients were studied using the Abbott Architect cTnI assay and the H-FABP assay. Special attention was paid to the diagnostic and prognostic value of admission blood samples taken <24 h after symptom onset. The prognostic endpoint was total mortality and reinfarction at 6 months. RESULTS: To detect forthcoming myocardial injury, admission samples gave receiver operating curve (ROC) areas (AUC) of 0.908 for cTnI and 0.855 for H-FABP (p=0.068) when the delay from symptom onset was <6 h (60.4% of all patients). When the delay was 6-24 h, the corresponding AUC values were 0.995 for cTnI and 0.849 for H-FABP (p=0.002). In multivariate analysis cTnI but not H-FABP predicted adverse events in all 293 patients (RR 3.02, 95% CI 1.62-5.63) and in those with delays <6 h (RR 2.92, 95% CI 1.47-5.81). CONCLUSION: In the era of highly sensitive cTnI assays, H-FABP appears to give no additional information even in patients who present within the first 6 h after acute MI.

Clinical significance of troponin I efflux and troponin autoantibodies in patients with dilated cardiomyopathy.

BACKGROUND: The appearance of circulating autoantibodies against cardiac troponin I (cTnAbs) in patients with heart failure has been reported. We sought to evaluate the role of circulating cardiac troponin I (cTnI) and cTnAbs in the pathophysiology and prognosis of idiopathic dilated cardiomyopathy. METHODS AND RESULTS: Circulating concentrations of cTnI and the presence of cTnAbs were determined in 95 patients with idiopathic dilated cardiomyopathy. The patients underwent laboratory testing, echocardiography, cardiopulmonary exercise testing, gated single photon emission computed tomography, and both-sided cardiac catheterization during a 3-day study period. Compared with cTnI- patients, the hearts of cTnI+ patients (cTnI > or = 0.01 ng/mL, n = 19) were significantly more dilated (left ventricular end-diastolic diameter 67 vs 61 mm, P < .05; left ventricular end-systolic dimension, 55 vs 49 mm, P < .01; echocardiography) and demonstrated greater intracardiac volumes (left ventricular end-diastolic volume 161 vs 132 mL, P = .060; left ventricular end-systolic volume 112 vs 82 mL, P < .05; gated single photon emission computed tomography), more disturbed systolic (ejection fraction 27 vs 33%, P < .05; gated single photon emission computed tomography) and cardiac sympathetic (123I-metaiodobenzylguanidine washout: 41% vs 34%; P < .05) function, and higher levels of vasoactive peptides (N-terminal proatrial natriuretic peptide 1030 vs 558 pmol/L, P < .05; N-terminal pro-B type natriuretic peptide 337 vs 115 pmol/L, P < .05). In addition, during a median follow-up time of 4.1 years, cTnI+ patients had clinical end points (cardiovascular death, heart transplantation, or clinical need for an automatic implantable cardioverter defibrillator) more often than cTnI- patients (37% vs 8%, P < .01). The presence of circulating cTnAbs (n = 15) was not associated with patients' clinical status or outcome. CONCLUSION: Patients with idiopathic dilated cardiomyopathy with cTnI efflux demonstrate more prominent changes in the indices of left ventricular remodeling and function than patients without signs of cTnI efflux. Moreover, elevated serum cTnI is associated with poor clinical outcome. The presence of circulating cTnAbs seems to have less utility in the clinical assessment of these patients. However, their pathogenic role in disease progression in the long term cannot be excluded.

Sensitive protein detection via triple-binder proximity ligation assays.

The detection of weakly expressed proteins and protein complexes in biological samples represents a fundamental challenge. We have developed a new proximity-ligation strategy named 3PLA that uses three recognition events for the highly specific and sensitive detection of as little as a hundred molecules of the vascular endothelial growth factor (VEGF), the biomarkers troponin I, and prostate-specific antigen (PSA) alone or in complex with an inhibitor--demonstrating the versatility of 3PLA.

Present and future biochemical markers for detection of acute coronary syndrome.

The use of biochemical markers in the diagnosis and management of patients with acute coronary syndrome has increased continually in recent decades. The development of highly sensitive and cardiac-specific troponin assays has changed the view on diagnosis of myocardial infarction and also extended the role of biochemical markers of necrosis into risk stratification and guidance for treatment. The consensus definition of myocardial infarction places increased emphasis on cardiac marker testing, with cardiac troponin replacing creatine kinase MB as the "gold standard" for diagnosis of myocardial infarction. Along with advances in the use of more cardiac-specific markers of myocardial necrosis, biochemical markers that are involved in the progression of atherosclerotic plaques to the vulnerable state or that signal the presence of vulnerable plaques have recently been identified. These markers have variable abilities to predict the risk of an individual for acute coronary syndrome. The aim of this review is to provide an overview of the well-established markers of myocardial necrosis, with a special focus on cardiac troponin I, together with a summary of some of the potential future markers of inflammation, plaque instability, and ischemia.

Utilization of recombinant Fab fragments in a cTnI immunoassay conducted in spot wells.

OBJECTIVES: To evaluate the performance of a new cTnI immunoassay utilizing site-specifically biotinylated recombinant Fab fragments on recently established spot wells. DESIGN AND METHODS: Two different cTnI-specific recombinant site-specifically biotinylated Fab fragments were produced. The performance of the new sandwich-type cTnI immunoassay in spot wells was evaluated in terms of binding capacity, assay kinetics and assay sensitivity and compared with a cTnI immunoassay carried out in conventional microtitration wells. Furthermore, the functionality of the recombinant Fab fragments was compared to the corresponding monoclonal antibodies in assay with one, two or three capture antibodies. RESULTS: The signal-to-background level was improved, providing an analytical detection limit of 0.002 microg/l with a surface of two capture Fab fragments. The spot wells increased the signal levels 2-fold and a further 4-fold improvement was detected with the Fab fragments already after 5 min assay time. CONCLUSIONS: The spot-concept in combination with site-oriented capture Fab fragments carries great promise as a very useful approach to improve the immunoassay performance of future point-of-care cTnI assays.

Comparison of cardiac troponin I immunoassays variably affected by circulating autoantibodies.

BACKGROUND: We recently provided evidence that circulating autoantibodies against cardiac troponin I (cTnI) or the troponin complex cause negative interference in cTnI immunoassays. By comparing three cTnI immunoassays, we further explored the phenomenon of circulating autoantibodies and their consequences in patient samples. METHODS: We developed a cTnI immunoassay with a novel assay design using three antibodies, two of which bind epitopes outside the stable, central part of cTnI. Samples from 541 chest pain patients were measured with the new cTnI assay and with a first-generation cTnI assay (Innotrac Aio cTnI) using a conventional midfragment assay design. Using another sample cohort, we also compared the new assay with a second-generation cTnI assay (Access AccuTnI). RESULTS: The analytical detection limit of the new cTnI assay was 0.012 microg/L, and the lowest concentration giving a total imprecision (CV) of 10% was 0.060 microg/L. The mean difference (95% limits of agreement) between the new cTnI and Aio cTnI assays was larger in admission samples (21.0%; -107.8% to 149.7%) than in samples taken 6-12 h (12.8%; -61.5% to 87.2%) and 24 h after admission (3.0%; -71.3% to 77.4%; P <0.001). With the lowest concentrations giving 10% CV (0.22 microg/L for Aio cTnI) used as cutoffs, 14.3% (n = 76) of admission samples were positive only with the new assay, whereas 13.5% (n = 72) were positive with both assays. Of samples taken at 6-12 and 24 h, 10.2% (n = 31) and 8.3% (n = 29) were positive only with the new assay. ROC curve analysis of admission samples showed a significantly higher area under the curve for the new cTnI assay (0.940) than for the Aio cTnI assay (0.846; P <0.001). The new cTnI assay gave generally lower results than the AccuTnI assay; the mean (95% limits of agreement) differences were -58.9% (-151.8% to 34.0%) in admission samples. In samples with severe interference from autoantibodies, median ratios between the new assay and AccuTnI were higher than in samples with no apparent troponin autoantibodies (0.875 vs 0.481; P<0.001). CONCLUSIONS: The new cTnI assay, which is based on a novel antibody combination different from the conventional midfragment antibody approach, offers improved detection of cTnI in samples containing troponin autoantibodies.

Negative interference in cardiac troponin I immunoassays by circulating troponin autoantibodies.

BACKGROUND: There are numerous potential sources of interference in immunoassays. Our aim was to identify the blood component that causes negative interference in cardiac troponin I (cTnI) immunoassays based on antibodies against the central part of cTnI. METHODS: We isolated an interfering factor (IF) from a sample with low recovery of added cTnI, using several consecutive purification steps: caprylic acid precipitation, ammonium sulfate precipitation, and purification on Cibacron Blue gel and protein G columns. Purified IF was identified by gel electrophoresis and mass spectrometric analysis of protein bands. For the direct detection of human antibodies to cardiac troponin in serum samples, we developed immunoassays using three different anti-human immunoglobulin antibodies and measured troponin antibodies in samples with low and normal cTnI recovery. RESULTS: Treatment with caprylic acid did not precipitate IF, but IF precipitated at 40% ammonium sulfate saturation. IF bound to a Cibacron Blue gel column, from which it was eluted with a linear salt gradient; it also bound to protein G. Gel electrophoresis of purified IF showed two major bands with molecular masses corresponding to the heavy (approximately 50 kDa) and light chains (approximately 25 kDa) of immunoglobulin, and their identities were confirmed by mass spectrometry. The presence of troponin-specific autoantibodies was confirmed in samples with low recoveries of cTnI by three different immunoassays. The median signals were significantly higher in 10 samples with low recovery than in 10 samples with normal recovery of cTnI (P < or = 0.007). CONCLUSIONS: Circulating autoantibodies to cTnI or other proteins of the troponin complex can be a source of negative interference in cTnI immunoassays.

Development of sensitive immunoassays for free and total human glandular kallikrein 2.

BACKGROUND: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostate-specific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity. METHODS: PSA- and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer. RESULTS: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 microg/L. The median total hK2 concentration was 0.022 microg/L (range, 0.0015-0.37 microg/L). hK2 concentrations were 0.1-58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41-50 and 51-60 years of age. The ratio of hK2 to PSA steadily decreased from 5-30% at PSA <1 microg/L to 1-2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 microg/L (range, 0.0015-16.2 microg/L). The median free hK2 concentration was 0.070 (range, 0.005-12.2) microg/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%). CONCLUSIONS: The wide variation in the free-to-total hK2 ratio suggests that hK2 in blood plasma is not consistently in the free, noncomplexed form in patients with prostate cancer. The new assay is sufficiently sensitive to be used to study the diagnostic accuracies of free and total hK2 for prostate cancer.

An interfering component in cardiac troponin I immunoassays-Its nature and inhibiting effect on the binding of antibodies against different epitopes.

OBJECTIVES: We recently reported on the occurrence of a common interfering factor (IF) that negatively affects determinations of cardiac troponin I (cTnI). The aim of the present investigation was to extend our initial finding by a detailed epitope-based determination of the location of IF and to reveal the approximate size and characteristics of IF. DESIGN AND METHODS: Two-site immunoassays using combinations of 16 monoclonal and 2 polyclonal cTnI antibodies and 1 monoclonal troponin C (TnC) antibody were used to measure the analytical recovery of cTnI or cTnI-TnC in serum samples. Gel filtration of serum samples containing IF was performed and the analytical recovery of cTnI in the fractions was determined. EDTA-plasma samples to which cTnI had been added and serum samples containing endogenous cTnI were also separated by gel filtration. RESULTS: The mean analytical recoveries of cTnI were 28.3% (range 7.5-55.1%) and of cTnI-TnC were 23.5% (range 8.7-51.8%) in samples containing IF when antibodies against midfragment epitopes of cTnI were used. The mean recovery of cTnI was 65.1% and 73.3% for antibodies with N- and C-terminal epitopes. Gel filtration of samples with low recovery of cTnI showed that the approximate molecular mass of IF was 50-200 kDa and that the cTnI elution profiles of samples containing IF were shifted towards higher molecular mass compared with samples with less IF. CONCLUSIONS: Antibodies against midfragment epitopes of cTnI are affected by IF to a considerable but variable extent, and the presence of IF can be demonstrated by gel filtration.

Negative interference in cardiac troponin I immunoassays from a frequently occurring serum and plasma component.

BACKGROUND: Cardiac troponin I (cTnI) is a sensitive marker of cardiac injury, but cTnI assays, like other immunoassays, are susceptible to interferences. We evaluated the presence of interfering substances by measuring the recovery of cTnI added to samples from volunteers and from patients with acute coronary syndromes (ACS). METHODS: We added a ternary complex of human cardiac troponin (30-500 microg/L) or cTnI from serum to samples from healthy volunteers and ACS patients. We measured cTnI with a two-site sandwich time-resolved immunofluorometric assay using two antibodies against epitopes in the central stable part of cTnI. We also analyzed 108 heparin-plasma samples from 16 ACS patients with this assay, with an assay based on four antibodies, and with two commercial cTnI assays, AxSYM and ACS:180. RESULTS: In samples from both healthy persons and ACS patients, recoveries for our assay were 1-167% (range). Recoveries were increased by addition of an antibody with an epitope in the N-terminal region of cTnI to the solid phase and an antibody with an epitope in the C-terminal region as a second detection antibody. In 2 of 16 patients with ACS, normal cTnI concentrations found when measured with the original assay demonstrated clinically abnormal (up to 10-fold higher) results with the additional N- and C-terminal antibodies in the early phase of infarction. Both commercial cTnI assays also demonstrated clinically misleading, falsely low cTnI concentrations. CONCLUSIONS: Some yet unidentified, variable component, present in the blood from healthy volunteers and ACS patients, interferes with the binding of antibodies against epitopes in the central part of cTnI used in two commercial assays. Our approach to supplement the mid-fragment cTnI antibodies with antibodies in the N- and C-terminal parts of the molecule in an experimental assay represents a step in resolving this interferent.

Release patterns of pregnancy associated plasma protein A (PAPP-A) in patients with acute coronary syndromes.

OBJECTIVE: Pregnancy associated plasma protein A (PAPP-A) has recently been shown to be associated with acute coronary syndromes (ACS). The goal of this study was to investigate its release patterns in patients with ACS. DESIGN: PAPP-A concentrations in plasma samples serially collected after admissions from 15 patients with ACS were measured. The levels of PAPP-A were compared with a reference range determined from 80 normal subjects. The associations between PAPP-A and myoglobin (Mb), C-reactive protein (CRP), fatty-acid-binding protein (FABP) and creatine kinase MB (CK-MB) were determined. RESULTS: Various release patterns were observed with 2-10-fold changes of PAPP-A in the different patients. Increases in PAPP-A levels above the reference range could appear early at 2 h or late at 30 h after onset of chest pain. Only in 4 of the 15 cases were significantly elevated PAPP-A levels detected before 6 h after onset. Elevations early after admission showed rapid decline whereas later elevations were more persistent. No associations between PAPP-A and Mb, CRP, FABP and CK-MB were found. However, a weak but significant association to cardiac troponin I (cTn I) was found. CONCLUSION: PAPP-A is an additional marker for ACS, but does not seem to be a useful early marker for acute myocardial infarction (AMI). The possible clinical utility of PAPP-A calls for extensive studies of chest pain patients using serial sampling combined with short- and long-term outcome studies.