Search Results (20)

Immunoassays for cardiac troponin, such as the Elecsys^® hs-TnT, have become the gold standard for myocardial infarction diagnostics. While various protein/chemical factors affecting the troponin complex and thus its diagnostic accuracy have been investigated, the role of coding single-nucleotide polymorphisms remains underexplored. To evaluate potential cSNP-induced interference with antibody binding in the Elecsys^® hs-TnT immunoassay, we applied ITEM-FOUR, a mass spectrometry-based method that quantifies changes in antibody binding upon amino acid substitutions in epitope peptides. Candidate cSNPs were selected from the dbSNP database and were mapped to human cardiac troponin T by molecular modeling. Consuming micromolar antibody concentrations and microliter sample volumes, two wild-type and 17 cSNP-derived variant epitope peptides—six for monoclonal antibody M7 and eleven for monoclonal antibody M11.7—were investigated to reveal the binding motifs “V₁₃₁-K₁₃₄-E₁₃₈-A₁₄₂” for M7 and “E₁₄₆-I₁₅₀-R₁₅₄-E₁₅₇” for M11.7. Loss of binding to M11.7 was observed for substitutions Q148R (rs730880232), R154W (rs483352832), and R154Q (rs745632066), whereas the E138K (rs730881100) exchange disrupted binding of M7. Except for cSNP Q148R, they are associated with cardiomyopathies, placing affected individuals at risk of both underlying heart disease and false-negative hs-TnT assay results in cases of myocardial infarction. Our results highlight the need to account for cSNP-related interferences in antibody-based diagnostics. ITEM-FOUR offers a powerful approach for tackling this challenge, fostering next-generation assay development. Full article

Background: International guidelines recommend the use of high-sensitivity cardiac troponin (hs-cTn) I and T methods for the detection of myocardial injury as a pre-requisite for the diagnosis of acute myocardial infarction (AMI) in patients admitted to the emergency department. Recently, Mindray (Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) has introduced a new chemiluminescence immunoassay (CLIA) for the detection of the cTn complex. The present study aims to verify and validate the hs-cTnI Mindray assay on the new automated CL2600i analyzer compared to the routine Alinity-i series instrument by Abbott (Abbott, Chicago, IL, USA). Methods: This study evaluated linearity, precision through the 5 × 5 protocol, methodological comparison on plasma and serum matrices, hs-cTnI 99th percentile imprecision, and the hs-cTnI detection rate in a healthy population. Results: The results obtained proved that the performance of the Mindray hs-cTnI test on the CL2600i platform was closely comparable to the Abbott Alinity-i system (plasma R²: 0.974; serum R²: 0.995). The CVs were consistently low, and no significant differences were reported. Excellent analytical performance, with high sensitivity, was also observed in the healthy population (overall detection rate: 79%), as well as good linearity within the measuring range (R²: 0.994). Conclusions: The Mindray hs-cTnI test confirms its robustness and utility in routine practice as an advanced assay. The new technology, with more sensitive detection methods, may improve the accuracy and reliability of cardiac biomarker testing, ultimately leading to better outcomes in the management of patients with AMI and other cardiac conditions. Full article

Cardiac troponin-I (cTnI) is a highly sensitive and specific marker of myocardial injury detectable in plasma by immunoassay techniques. Inclusion criteria over a 3-year period required a diagnosis of cardiac disease accompanied by electrocardiographic (ECG) and cardiac ultrasound examinations (n = 23) in adult horses (≥2 years of age). A second group of normal adult ponies (n = 12) was studied as a reference group. Heparinized jugular venous blood samples were collected and centrifuged within 30 min, and the plasma was separated and frozen at −70 °C for subsequent batched cTnI analysis. The lower limit of detection was 0.01 ng/mL, and the upper limit was 100 ng/mL of plasma. Normal equine plasma cTnI concentrations ranged from 0.01 to 0.03 ng/mL (n = 12). Horses with non-arrhythmogenic murmurs (n = 4) included tricuspid (0.05 ng/mL cTnI), mitral (0.07), and aortic insufficiencies (0.01, 0.02). Horses with benign atrial fibrillation (n = 8) had a cTnI range of <0.01–0.09 ng/mL, with four horses having cTnI concentrations falling slightly outside the reference range (0.04, 0.05, 0.06, and 0.09). Horses with ventricular arrhythmias (ventricular premature contractions or ventricular tachycardia) and documentable myocardial toxicities or immunological reactions (n = 5) had cTnI concentrations of 0.05, 0.21, 0.31, 15.18, and >100 ng/mL. Horses with ventricular arrhythmias but no documentation of myocardial toxicity (n = 3) had cTnI concentrations of 0.34, 0.46, and 80.42 ng/mL. When grouped by arrhythmia type and compared using the Mann–Whitney Rank Sum Test, the median ventricular arrhythmia cTnI (0.40 ng/mL) was significantly higher than the median atrial fibrillation cTnI (0.04 ng/mL, p < 0.001). It was concluded that horses with myocardial toxicities and ventricular arrhythmias often had severe elevations in plasma cTnI. Full article

The measurement of cardiac troponin I (cTnI) is of vital importance for the early diagnosis of acute myocardial infarction. In this study, an enhanced electrochemiluminescent immunoassay for the highly sensitive and precise determination of cTnI was reported. A biomimetic chip with nepenthes peristome surface microstructures to achieve single-layer microbead arrays and integrated microelectrode arrays (MEAs) for ECL detection was microfabricated. Ru@SiO₂ nanoparticles were prepared as signal amplificators labeling immunomagnetic beads. Dendrimer-encapsulated platinum nanoparticles (Pt DENs) were electrochemically modified on ITO MEAs. The resulting Pt DEN-modified ITO MEAs preserved good optical transparency and exhibited an approximately 20-fold ECL signal amplification compared to that obtained from bare ITO. The method made full use of the biomimetic chip with Pt DENs to develop single-layer immunomagnetic bead arrays with increasingly catalyzed electrochemical oxidation of the [Ru(bpy)₃]²⁺–TPA system. Consequently, a limit of detection calculated as 0.38 pg/mL (S/N = 3) was obtained with excellent selectivity, demonstrating significant potential for the detection of cTnI in clinical diagnostics. Full article

The myocardial infarction (MI) types 4a and 5 guidelines recommend cardiac troponin (cTn) diagnostic decision limits of 5 and 10 times the 99th percentile, respectively. Different cTn kits elicit different responses, so the MI diagnosis is still challenging. The study aimed to establish the cutoff values and the accuracy of three different cTnI kits in the diagnosis of post-procedural MI. We analyzed 115 patients with multivessel stable chronic coronary artery disease; 26 underwent percutaneous coronary intervention, and 89 underwent coronary artery bypass graft. Delayed-enhancement magnetic resonance imaging was performed before and after each intervention for definitive MI diagnoses. Two contemporary and one high-sensitivity cTnI immunoassays were used. ROC curves determined the accuracy of each assay. Low accuracy was observed after applying the current guidelines recommendations. The three cTnI assays accuracies improved when adjusted by the new ROC cutoffs, reaching 82% for MI type 5 for all assays, and 78%, 88%, and 87% for MI type 4 for Siemens, Beckman, and Abbott, respectively. The ultrasensitive and contemporary tests’ accuracy for MI types 4a and 5 diagnoses are equivalent when adjusted for these new cutoffs. The hs-cTnI assays had lower accuracy than contemporary tests for MI types 4a and 5 diagnoses. Full article

Rapid and accurate detection of acute myocardial infarction can improve patients’ chances of survival. Cardiac troponin I (cTn I) is an important diagnostic biomarker for acute myocardial infarction. However, current immunoassays are insufficient to accurately measure cTn I, as they have limited detection sensitivity and are time-consuming. Surface-enhanced Raman scattering (SERS) is a brilliant fingerprints diagnostic technique characterised by ultrasensitivity, fast response, and qualitative and quantitative analysis capabilities. In this study, reporter molecules (4-Mercaptobenzoic acid, 4-MBA) embedded Au@Ag core-shell nanospheres as SERS nanotags were prepared for the detection of cTn I. As the Raman reporters were embedded between the core and the shell, they could be protected from the external environment and nanoparticle aggregation. Excellent SERS performances were obtained due to the enhanced local electromagnetic field in the gap of core and shell metals. In a standard phosphate buffered saline (PBS) environment, the limit of detection for cTn I was 0.0086 ng mL⁻¹ (8.6 ppt) with a good linear relationship. The excellent Raman detection performance was attributed to the localized surface plasmon resonance effect and strong electromagnetic field enhancement effect produced by the gap between the Au core and the Ag shell. The SERS nanotags we prepared were facile to synthesize, and the analysis procedure could be completed quickly (15 min), which made the detection of cTn I faster. Therefore, the proposed SERS nanotags have significant potential to be a faster and more accurate tool for acute myocardial infarction diagnostics. Full article

The aim of the present study was to investigate the levels of YKL-40 during and after coronary artery bypass grafting surgery (CABG) and to establish possible connections between YKL-40 and markers of oxidative stress, inflammation, and myocardial injury. Patients undergoing elective CABG utilizing cardiopulmonary bypass (CPB) were recruited into the study. Blood samples were collected at the onset of anesthesia, during surgery and post-operatively. Levels of YKL-40, 8-isoprostane, interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and troponin T (TnT) were measured by immunoassay. YKL-40 levels increased significantly 24 h after CPB. Positive correlation was seen between post-operative TnT and YKL-40 levels (r = 0.457, p = 0.016) and, interestingly, baseline YKL-40 predicted post-operative TnT increase (r = 0.374, p = 0.050). There was also a clear association between YKL-40 and the chemotactic factors MCP-1 (r = 0.440, p = 0.028) and IL-8 (r = 0.484, p = 0.011) linking YKL-40 to cardiac inflammation and fibrosis following CABG. The present results show, for the first time, that YKL-40 is associated with myocardial injury and leukocyte-activating factors following coronary artery bypass surgery. YKL-40 may be a factor and/or biomarker of myocardial inflammation and injury and subsequent fibrosis following heart surgery. Full article

Express and highly sensitive immunoassays for the quantitative registration of cardiac troponin I (cTnI) are in high demand for early point-of-care differential diagnosis of acute myocardial infarction. The selection of antibodies that feature rapid and tight binding with antigens is crucial for immunoassay rate and sensitivity. A method is presented for the selection of the most promising clones for advanced immunoassays via simultaneous characterization of interaction kinetics of different monoclonal antibodies (mAb) using a direct label-free method of multiplex spectral correlation interferometry. mAb-cTnI interactions were real-time registered on an epoxy-modified microarray glass sensor chip that did not require activation. The covalent immobilization of mAb microdots on its surface provided versatility, convenience, and virtually unlimited multiplexing potential. The kinetics of tracer antibody interaction with the “cTnI—capture antibody” complex was characterized. Algorithms are shown for excluding mutual competition of the tracer/capture antibodies and selecting the optimal pairs for different assay formats. Using the selected mAbs, a lateral flow assay was developed for rapid quantitative cTnI determination based on electronic detection of functionalized magnetic nanoparticles applied as labels (detection limit—0.08 ng/mL, dynamic range > 3 orders). The method can be extended to other molecular biomarkers for high-throughput screening of mAbs and rational development of immunoassays. Full article

Cardiac troponins (cTns) have long been the most valuable and specific biomarkers for detecting ischemic myocardial cells (MCs) injury, which is one of the key signs of myocardial infarction (MI). Modern methods (highly sensitive and ultra-sensitive immunoassays (hs-cTns)) of detection are an important and indispensable tool for the early diagnosis of MI and the choice of patient management protocols. Timely diagnosis of MI can significantly improve the prognosis of patients. However, in real clinical practice, doctors often face a significant problem when using cTns—the difficulty of differential diagnosis due to frequent and unexplained increases in the concentration of cTns in blood serum. In addition, there is conflicting information that may potentially affect the diagnostic capabilities and value of cTns: the influence of certain biological factors (diurnal rhythm, gender and age) on serum cTns levels; extra-cardiac expression of cTns; the possibilities of non-invasive diagnosis of MI; and other pathological conditions that cause non-ischemic injury to MCs. To solve these problems, it is necessary to concentrate on studying the metabolism of cTns. The review of our current knowledge about cTns metabolism consists of two parts. In this (first) part of the manuscript, the main stages of cTns metabolism are briefly described and the mechanisms of cTns release from MCs are considered in detail. Full article

Understanding of the biology of endo- and exogenous molecules, in particular their metabolism, is not only of great theoretical importance, but also of high practical significance, since many molecules serve as drug targets or markers for the laboratory diagnostics of many human diseases. Thus, cardiac troponin (cTns) molecules have long been used as key markers for the confirmation of diagnosis of myocardial infarction (MI), and with the introduction of contemporary (high sensitivity) test methods, many of our concepts related to the biology of these cardiac markers have changed significantly. In current clinical practice, there are opening new promising diagnostic capabilities of cTns, the understanding and justification of which is closely connected with the theoretical principles of the metabolism of these molecules. However, today, the biology and metabolism of cTns have not been properly investigated; in particular, we do not know the precise mechanisms of release of these molecules from the myocardial cells (MCs) of healthy people and the mechanisms of circulation, and the elimination of cTns from the bloodstream. The main purpose of this manuscript is to systematize information about the biology of cTns, with an emphasis on the metabolism of cTns. The format of this paper, starting with the release of cTns in the blood and concluding with the metabolism/filtration of troponins, provides a comprehensive yet logically easy way for the readers to approach our current knowledge in the framework of understanding the basic mechanisms by which cTns are produced and processed. Conclusions. Based on the analysis of the current literature, the important role of biology and all stages of metabolism (release, circulation, removal) of cTns in laboratory diagnostics should be noted. It is necessary to continue studying the biology and metabolism of cTns, because this will improve the differential diagnosis of MI and i a new application of cTns immunoassays in current clinical practice. Full article

The sensitivity of immunoassays was reported to be increased by the orientation of antibodies. We investigated how the size and valence of antigens and orientation and valence of antibodies contribute to the analytical sensitivity of ELISA. Antigens differing in size and number of epitopes were compared using oriented and non-oriented ELISAs: the orientation of antibodies was obtained coating half-fragment antibodies on maleimide microplates, while, in non-oriented ELISA, whole antibodies were randomly physisorbed. The oriented assay performed better than the non-oriented one at each concentration (0.4–3.3 ng/mL) of a small monomeric antigen (cardiac Troponin I, 24 kDa, Rh 3 nm). No significant differences were observed with a large monovalent antigen (prostate-specific antigen-alpha(1) antichymotrypsin, 90 kDa, Rh > 3 nm), since its steric hindrance overcame the increased availability of antigen binding sites given by orientation. Large multivalent antigens (ferritin, 280 kDa, Rh 6 nm; α-fetoprotein, >70 kDa, Rh > 3.3 nm) performed better in non-oriented assays. In this case, the repeated epitopes on the surface of the antigens favored the engagement of both antigen binding sites of the whole IgG, thus suggesting that avidity represented the leading force in this experimental setting. In conclusion, the design of high-sensitivity ELISAs should consider the dimension and valency of antigens in addition to the affinity and avidity of antibodies. Full article

Cardiovascular diseases (CVDs) are one of the foremost causes of mortality in intensive care units worldwide. The development of a rapid method to quantify cardiac troponin I (cTnI)—the gold-standard biomarker of myocardial infarction (MI) (or “heart attack”)—becomes crucial in the early diagnosis and treatment of myocardial infarction (MI). This study investigates the development of an efficient fluorescent “sandwich” immunoassay using liposome-based fluorescent signal amplification and thereby enables the sensing and quantification of serum-cTnI at a concentration relevant to clinical settings. The calcein-loaded liposomes were utilized as fluorescent nano vehicles, and these have exhibited appropriate stability and efficient fluorescent properties. The standardized assay was sensitive and selective towards cTnI in both physiological buffer solutions and spiked human serum samples. The novel assay presented noble analytical results with sound dynamic linearity over a wide concentration range of 0 to 320 ng/mL and a detection limit of 6.5 ng/mL for cTnI in the spiked human serum. Full article

Cardiovascular diseases are considered one of the major causes of human death globally. Myocardial infarction (MI), characterized by a diminished flow of blood to the heart, presents the highest rate of morbidity and mortality among all other cardiovascular diseases. These fatal effects have triggered the need for early diagnosis of appropriate biomarkers so that countermeasures can be taken. Cardiac troponin, the central key element of muscle regulation and contraction, is the most specific biomarker for cardiac injury and is considered the “gold standard”. Due to its high specificity, the measurement of cardiac troponin levels has become the predominant indicator of MI. Various forms of diagnostic methods have been developed so far, including chemiluminescence, fluorescence immunoassay, enzyme-linked immunosorbent assay, surface plasmon resonance, electrical detection, and colorimetric protein assays. However, fluorescence-based immunoassays are considered fast, accurate and most sensitive of all in the determination of cardiac troponins post-MI. This review represents the strategies, methods and levels of detection involved in the reported fluorescence-based immunoassays for the detection of cardiac troponin I. Full article

This work demonstrates the quantitative assay of cardiac Troponin I (cTnI), one of the key biomarkers for acute cardiovascular diseases (the leading cause of death worldwide) using the fluorescence-based sandwich immune reaction. Surface plasmon coupled emission (SPCE) produced by non-radiative coupling of dye molecules with surface plasmons being excitable via the reverse Kretschmann format is exploited for fluorescence-based sandwich immunoassay for quantitative detection of cTnI. The SPCE fluorescence chip utilizes the gold (2 nm)-silver (50 nm) bimetallic thin film, with which molecules of the dye Alexa 488 (conjugated with detection antibodies) make a near field coupling with the plasmonic film for SPCE. The experimental results find that the SPCE greatly improves the sensitivity via enhancing the fluorescence signal (up to 50-fold) while suppressing the photo-bleaching, permitting markedly enhanced signal-to-noise ratio. The limit of detection of 21.2 ag mL⁻¹ (atto-gram mL⁻¹) is obtained, the lowest ever reported to date amid those achieved by optical technologies such as luminescence and label-free optical sensing techniques. The features discovered such as ultrahigh sensitivity may prompt the presented technologies to be applied for early diagnosis of cTnI in blood, particularly for emergency medical centers overloaded with patients with acute myocardial infarction who would suffer from time-delayed diagnosis due to insufficient assay device sensitivity. Full article

This paper presents a lateral flow assay (LFA) for the quantitative, fluorescence-based detection of the cardiac biomarker troponin I (cTnI) that features an analytical strip made of cellulose filter paper. The results show that the wicking and test time are comparable to those obtained with conventional nitrocellulose (NC)-based LFAs. Further, the cellulose paper provides an excellent background with no auto-fluorescence that is very adequate in detecting fluorescent lines. While fluorescence that was generated with cellulose strips was lower when compared to that generated in NC strips, signals could be improved by layering carbon nanofibers (CNF) on the cellulose. A nonlinear behavior of the concentration–response relationship was observed for the LFA architectures with NC, cellulose, and cellulose-CNF in the 0 to 200 ng/mL cTnI concentration range. The measurements were consistent and characterized by coefficients of variation lower than 2.5%. Detection and quantitation limits that were in the range 1.28–1.40 ng/mL and 2.10–2.75 ng/mL were obtained for LFA with cellulose and cellulose CNF strips that are equivalent to the limits obtained with the standard NC LFA. Overall, we showed that commercially available filter paper can be used in the analytical strip of LFA. Full article