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A novel immunoassay platform is presented utilizing a cardiac troponin T antibody (Ab-cTnT) labelled with 5-carboxyfluorescein (5-FAM) integrated into a two-dimensional (2D) manganese dioxide nanosheet (MnO 2 NS) matrix. This strategy enables a turn-on response towards cTnT antigen within a mere 10-min incubation period, boasting an impressive lower detection limit of 0.038 ng/mL. Crucially, our probe demonstrates exceptional selectivity amidst the presence of coexisting biomolecules and ions, ensuring precise detection of cTnT. Moreover, the developed platform showcases promising utility in sensing cTnT from spiked human serum samples, yielding satisfactory recovery percentages ranging from 82 to 105%. Additionally, we introduce and easy-to-use and cost-effective test strip for point-of-care detection of cTnT, further enhancing accessibility to critical cardiovascular diagnostics., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A "turn-on" photon up conversion nano couple based on NaYF 4 : Yb, Tm UCNPs quenched with MnO 2 nanosheet was developed for the rapid and selective detection of cTnT. Herein, MnO 2 nanosheet hold on the surface of Antibody cTnT (Ab-cTnT) conjugated blue emitting up conversion nanoprobe (λ em at 475 nm), which leads to quenching of fluorescence due to energy transfer from photon up conversion nanoparticles to MnO 2 nanosheets. On introducing cTnT antigen to the system, the energy transfer process is hindered due to strong antigen -antibody interface on the surface. This in turn, influences the nano-couples positions and effectively separates up conversion nanoprobe from MnO 2 nanosheets surface resulting in restriction to energy transfer process enabling fluorescence recovery. The developed probe shows a linear response towards cTnT in the range of 0.16-2.77 ng/mL with a Limit of Detection (LoD) of 0.025 ng/mL. The practical feasibility of the nanoprobe is performed with possible coexisting biomolecules. Biological study in human blood serum samples exhibited sufficient recovery percentage in the range of 92-103 % is obtained., Competing Interests: Declaration of competing interest The author declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cardiovascular disease has remained the world's biggest killer for 30 years. To aid in the diagnosis and prognosis of patients suffering cardiovascular-related disease accurate detection methods are essential. For over 20 years, the cardiac-specific troponins, I (cTnI) and T (cTnT), have acted as sensitive and specific biomarkers to assist in the diagnosis of various types of heart diseases. Various cardiovascular complications were commonly detected in patients with COVID-19, where cTn elevation is detectable, which suggested potential prognostic value of cTn in COVID-19-infected patients. Detection of these biomarkers circulating in the bloodstream is generally facilitated by immunoassays employing cTnI- and/or cTnT-specific antibodies. While several anti-troponin assays are commercially available, there are still obstacles to overcome to achieve optimal troponin detection. Such obstacles include the proteolytic degradation of N and C terminals on cTnI, epitope occlusion of troponin binding-sites by the cTnI/cTnT complex, cross reactivity of antibodies with skeletal troponins or assay interference caused by human anti-species antibodies. Therefore, further research into multi-antibody based platforms, multi-epitope targeting and rigorous validation of immunoassays is required to ensure accurate measurements. Moreover, in combination with various technical advances (e.g. microfluidics), antibody-based troponin detection systems can be more sensitive and rapid for incorporation into portable biosensor systems to be used at point-of care., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objectives: Macrotroponin is due to cardiac troponin (cTn) binding to endogenous cTn autoantibodies. While previous studies showed a high incidence of macrotroponin affecting cTnI assays, reports of macrotroponin T, particularly without cTnI reactivity, have been rare. Although the clinical significance of macrotroponin is not fully understood, macroenzymes and complexes are recognised to cause confusion in interpretation of laboratory results. The potential for adverse clinical consequences due to misinterpretation of affected results is very high., Methods: We describe four cases of macrotroponin T with persistently low high sensitivity cTnT (hs-cTnT) by the 9 min compared to the 18 min variant of the assay. Three cases were serendipitously identified due to the use of a lot number of Roche hs-cTnT affected by non-reproducible results, necessitating measurement of cTnT in duplicate. We identified and characterised these macrotroponin specimens by immunoglobulin depletion (Protein A and PEG precipitation), mixing studies with EDTA and recombinant cTnT., Results: In cases of macro-cTnT, a lower result occurred on the hs-cTnT using the 9 min compared to 18 min variant assay (ratio of 9-18 min hs-cTnT <0.80). Mixing studies with recombinant cTnT or EDTA demonstrated a difference in recovery vs. controls. One of these patients demonstrated a high molecular weight complex for cTnI and cTnT demonstrating a macrocomplex involving both cTn. This patient demonstrated a rise and fall in cTn when measured by several commercial assays consistent with genuine acute cardiac injury., Conclusions: We identified several cases of macro-cTnT and described associated clinical and biochemical features., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Highly fluorescent hollow ZrO 2 @CdTe nanoparticles (NPs) were synthesized efficiently via the hydrothermal method. By changing the hydrothermal time of ZrO 2 @CdTe NP, the peaks of fluorescence spectra measured at fluorescent excitation of 330 nm were at 540 nm, 590 nm, and 640 nm, respectively. Hollow ZrO 2 NPs have a uniform core-shell structure with the size of 178 ± 10 nm and shell of 19 ± 4 nm. The as-prepared yellow-ZrO 2 @CdTe NPs were used to develop lateral flow assay (LFA) for the sensitive and qualitative detection of C-reactive protein (CRP). The visual limit of detection of the LFA for the CRP antigen was 1 μg/L within 20 min, which is 1000-fold lower than that of colloidal gold-based LFA. In addition, a multiplex lateral flow assay (mLFA) was developed using the as-prepared green and red-ZrO 2 @CdTe NPs for the simultaneous, specific, sensitive, and qualitative detection of CRP and troponin T (cTnT). The visual limits of detection of CRP and cTnT in mLFA were 10 μg/L and 0.1 mg/L, respectively. The excellent performance of ZrO 2 @CdTe NPs should facilitate their application in point-of-care technology for the detection of other biomarkers.

This study investigated the function of Troponin T (TnT) in the mud crab, Scylla paramamosain. The 1952 bp cDNA sequence of TnT was cloned from S. paramamosain using rapid amplification of cDNA ends (RACE) PCR. The quantitative real-time PCR analysis showed that TnT was highly expressed in the muscle and heart of S. paramamosain. Challenging with white spot syndrome virus (WSSV) or Vibrio alginolyticus (VA), two common pathogens that infect mud crabs, enhanced the expression of TnT in S. paramamosain. Knockdown of TnT using TnT-dsRNA led to up-regulating the expression of immune-related genes, such as c-type-lectin, toll-like-receptor, crustin antimicrobial peptide and prophenoloxidase. The cumulative mortality of WSSV- and VA-infected crabs was significantly increased following TnT knockdown. After WSSV or VA infection, TnT knockdown caused a significant reduction in phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC), indicating a regulatory role of TnT in the innate immune response of S. paramamosain to pathogens. Apoptosis of hemocytes was higher in crabs treated with TnT-dsRNA compared with control crabs treated with phosphate-buffered saline. Knockdown of TnT increased apoptosis of hemocytes following VA infection, but reduced hemocyte apoptosis following WSSV infection. In summary, TnT may enhance the immune response of S. paramamosain to WSSV infection by regulating apoptosis, THC, PO activity and SOD activity. And TnT may play a positive role in the immune response against VA infection by regulating apoptosis, THC, SOD activity and PO activity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Paper-based analytical devices (PADs) are widely used in point-of-care testing (POCT) as they are cost-effective, simple and straightforward. However, poor sensitivity hinders their use in detecting diseases with low abundance biomarkers. The poor detection limit of PADs is mainly attributed to the low concentration of analytes, and the complexity of biological fluid, leading to insufficient interactions between analytes and capture antibodies. This study aims to overcome these difficulties by developing a paper-based cationic isotachophoresis (ITP) approach for simultaneously detecting pico-molar levels of two essential cardiac protein markers: acidic troponin T (cTnT) and basic troponin I (cTnI) spiked into human serum samples. The approach utilizes 3-aminopropyltrimethoxysilane (APTMS) treated glass fiber papers with decreasing cross-sectional area assembled on a 3D printed cartridge device. Our results showed that in the presence of cTnT monoclonal antibody (mAb), fluorescently labeled cTnI and cTnT could be effectively enriched in cationic ITP. Each individual target was captured subsequently by a test line in the detection zone where the capture mAb was immobilized. Detailed analysis suggests that the technology is capable of simultaneous on-board depletion of abundant plasma proteins and enrichment of cTnI/cTnT by ~1300-fold with a sensitivity of 0.6 pmol/L for cTnT and a sensitivity of 1.5 pmol/L for cTnI in less than 6 min. The results demonstrate the potential of this technology for rapid, ultra-sensitive and cost-effective analysis of multiplex protein markers in clinical serum samples at point of care., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Inflammation is involved in initiation and progression of aortic stenosis (AS). However, the role of the complement system, a crucial component of innate immunity in AS, is unclear. We hypothesized that circulating levels of complement factor B (FB), an important component of the alternative pathway, are upregulated and could predict outcome in patients with severe symptomatic AS. Therefore, plasma levels of FB, Bb, and terminal complement complex were analyzed in three cohorts of patients with severe symptomatic AS and mild-to-moderate or severe asymptomatic AS (population 1, n = 123; population 2, n = 436; population 3, n = 61) and in healthy controls by enzyme immunoassays. Compared with controls, symptomatic AS patients had significantly elevated levels of FB (2.9- and 2.8-fold increase in population 1 and 2, respectively). FB levels in symptomatic and asymptomatic AS patients were comparable (population 2 and 3), and in asymptomatic patients FB correlated inversely with valve area. FB levels in population 1 and 2 correlated with terminal complement complex levels and measures of systemic inflammation (i.e., CRP), cardiac function (i.e., NT-proBNP), and cardiac necrosis (i.e., Troponin T). High FB levels were significantly associated with mortality also after adjusting for clinical and biochemical covariates (hazard ratio 1.37; p = 0.028, population 2). Plasma levels of the Bb fragment showed a similar pattern in relation to mortality. We concluded that elevated levels of FB and Bb are associated with adverse outcome in patients with symptomatic AS. Increased levels of FB in asymptomatic patients suggest the involvement of FB from the early phase of the disease., (Copyright © 2019 by The American Association of Immunologists, Inc.)

The authors describe a method for the improvement of the sensitivity of immunoassays. This was achieved by a combination of immunoassay and an amplification based on the use of a multiplied DNA probe. The immunoassay was enhanced via recombinant polymerase amplification (RPA). It enables fast (35 min) isothermal multiplication of DNA at 37 °C. This concept was demonstrated for a sandwich immunoassay that is making use of magnetic nanoparticles conjugated to first specific antibodies and then to second specific antibodies linked to reporter DNA via biotin-streptavidin binding. Reporter DNA multiplied by RPA was quantified fluorometrically via the carboxyfluorescein label. Human cardiac troponin T (cTnT), a biomarker for acute myocardial infarction, was used as the target of the immunoRPA (iRPA). The assay can detect cTnT in serum and in plasma within 2 h, with detection limits as low as 12.5 ± 1.1 pg•mL -1 and 9.4 ± 2.1 pg•mL -1 , respectively. This represents increased sensitivity and decreased assay time compared to classical ELISAs (3.5 ± 0.3 ng•mL -1 ) or immuno-PCR (4.3 ± 0.8 ng•mL -1 ) that were carried out using the same immunoreagents and reporter DNA. The good performance of this iRPA was underlined by its successful application to the analysis of plasma samples. The iRPA has significant advantages relative to other DNA amplification-based immunoassays due to isothermal conditions and analysis at 37 °C. Graphical abstract Schematic representation of a new kind of immunoassay that is enhanced by making use of recombinase polymerase amplification (immunoRPA). Reporter DNA was conjugated with specific antibodies, then amplified and finally quantified fluorometrically. Limit of detection of troponin T in plasma within 2 h was 9.4 ± 2.1 pg•mL -1 .

Cardiac dysfunction, a common consequence of sepsis, is the major contribution to morbidity and mortality in patients. Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of Tanshinone IIA (TA), a main active component of Salvia miltiorrhiza Bunge, which has been widely used in China for the treatment of cardiovascular and cerebral system diseases. In the present study, the effect of STS on sepsis-induced cardiac dysfunction was investigated and its effect on survival rate of rats with sepsis was also evaluated. STS treatment could significantly decrease the serum levels of C-reactive protein (CRP), procalcitonin (PCT), cardiac troponin I (cTn-I), cardiac troponin T (cTn-T), and brain natriuretic peptide (BNP) in cecal ligation and puncture (CLP)-induced) septic rats and improve left ventricular function, particularly at 48 and 72 h after CLP. As the pathogenesis of septic myocardial dysfunction is attributable to dysregulated systemic inflammatory responses, several key cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10) and high mobility group protein B1 (HMGB1), were detected to reveal the possible mechanism of attenuation of septic myocardial dysfunction after being treated by STS. Our study showed that STS, especially at a high dose (15 mg·kg -1 ), could efficiently suppress inflammatory responses in myocardium and reduce myocardial necrosis through markedly reducing production of myocardial TNF-α, IL-6 and HMGB1. STS significantly improved the 18-day survival rate of rats with sepsis from 0% to 30% (P < 0.05). Therefore, STS could suppress inflammatory responses and improve left ventricular function in rats with sepsis, suggesting that it may be developed for the treatment of sepsis., (Copyright © 2018 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

In this study, we demonstrated a prompt and sensitive detection technique for cardiac troponin T (cTnT) in buffer and biological fluid (serum) using an NIR-active fluorescent anti-cTnT-labelled carbon dot (CD) and molybdenum disulfide (MoS2)-based nano-couple. Exfoliated MoS2 nanosheets strongly grasp the anti-cTnT-labelled CDs over their surface, and an excited-state non-radiative energy transfer mechanism takes place from CDs to MoS2, thereby quenching the upconversion fluorescence. The nonlinear and upward Stern-Volmer relationship is observed, which indicates a combined static and dynamic quenching. Static and time-resolved fluorescence measurements predict distance-dependent Förster resonance energy transfer (FRET) dynamics, which control the detection process. In the presence of cTnT, the energy transfer process gets hindered due to strong antibody/antigen (anti-cTnT/cTnT) interaction. The cTnT molecules affect the positions of the nano-couple and cause effective detachment of CDs from the MoS2 surface. This results hindrance in the energy transfer process with consequent restoration of upconversion intensity. A linear response is observed between the cTnT concentration and the restored fluorescence intensity in the concentration range of 0.1-50 ng mL-1 with a limit of detection of 0.12 ng mL-1 and a limit of quantification of 0.38 ng mL-1. Statistical analysis shows that the present assay possesses an accuracy of 101.4 ± 3.76 with a co-relation co-efficient of 0.99. Thus, CD/MoS2 provides a promising platform for the sensitive detection of cTnT.

Regeneration of immunosensors is a longstanding challenge. We have developed a re-usable troponin-T (TnT) immunoassay based on localised surface plasmon resonance (LSPR) at gold nanorods (GNR). Thermosensitive poly(N-isopropylacrylamide) (PNIPAAM) was functionalised with anti-TnT to control the affinity interaction with TnT. The LSPR was extremely sensitive to the dielectric constant of the surrounding medium as modulated by antigen binding after 20 min incubation at 37 °C. Computational modelling incorporating molecular docking, molecular dynamics and free energy calculations was used to elucidate the interactions between the various subsystems namely, IgG-antibody (c.f., anti-TnT), PNIPAAM and/or TnT. This study demonstrates a remarkable temperature dependent immuno-interaction due to changes in the PNIPAAM secondary structures, i.e., globular and coil, at above or below the lower critical solution temperature (LCST). A series of concentrations of TnT were measured by correlating the λ LSPR shift with relative changes in extinction intensity at the distinct plasmonic maximum (i.e., 832 nm). The magnitude of the red shift in λ LSPR was nearly linear with increasing concentration of TnT, over the range 7.6 × 10 -15 to 9.1 × 10 -4  g/mL. The LSPR based nano-immunoassay could be simply regenerated by switching the polymer conformation and creating a gradient of microenvironments between the two states with a modest change in temperature.

Background: Autoantibodies to cardiac troponins (TnAAbs) could negatively affect cardiac troponin I (cTnI) measurements by TnAAbs-sensitive immunoassays. We investigated the epitope specificity of TnAAbs and its influence on cTnI immunodetection in patients with acute myocardial infarction (AMI)., Methods: The specificity of TnAAbs was studied in immunoassays and gel-filtration experiments. The influence of TnAAbs on endogenous troponin measurements was studied in 35 plasma samples from 15 patients with AMI., Results: The inhibitory effect of TnAAbs on the cTnI immunodetection was observed only for the ternary cardiac troponin complex (I-T-C) and not for the binary cardiac troponin complex (I-C) or free cTnI. In the same TnAAbs-containing samples, the immunodetection of cardiac troponin T (cTnT) added in the form of I-T-C (but not free cTnT) was also inhibited in the assays that used monoclonal antibodies (mAbs) specific to the 223-242 epitope. The negative effects of TnAAbs on the measurements of endogenous cTnI in AMI samples were less than on the measurements of isolated I-T-C and decreased with time after the onset of symptoms. Early AMI blood samples might contain a mixture of the I-T-C and I-C complexes with the ratio gradually changing with the progression of the disease in favor of I-C., Conclusions: The investigated TnAAbs are specific to the structural epitopes formed by cTnI and cTnT molecules in the I-T-C complex. AMI blood samples contain a mixture of I-C and I-T-C complexes. The concentrations of total cTnI at the early stage of AMI could be underestimated in approximately 5%-10% of patients if measured by TnAAbs-sensitive immunoassays., (© 2016 American Association for Clinical Chemistry.)

The aim of this study is to prepare and characterize cardiac troponin T (cTnT) monoclonal antibodies (mAb), and further develop a chemiluminescence quantitative detection assay for cTnT. BALB/c mice were immunized with recombinant cTnT antigen, and specific mAbs were prepared using conventional hybridoma technique and screened by indirect ELISA method. To identify the epitopes, several cTnT peptide fragments were synthesized or expressed by genetic engineering. A double antibody sandwich ELISA method was used to screen the mAb pairs for cTnT detection, and the automatic chemiluminescence detection assay for cTnT was developed. In total 220 clinical specimens were used for system comparison between our assay and Roche cTnT assay; further performance characteristics was evaluated by testing 238 clinical samples and 784 physical examination samples. We successfully screened 33 strains of hybridoms against cTnT, and the mAbs' epitopes were identified. Mab E16H8 and C8G11 with a detection limit of 10 pg/mL cTnT antigen were selected to develop the full automatic chemiluminescence quantitative assay. The correlation coefficient of our reagent with Roche's was 0.959 9, with a coincidence rate of 95%. The assay presented a sensitivity of 97.5%, and a specificity of 99.15% in detection of clinical samples. The cTnT concentration was less than 0.080 6 ng/mL in 99% of general population, which agrees with the definition of WHO on patients with acute myocardial infarction (AMI). In summary, we developed monoclonal antibodies against predominant epitopes for diagnostics of cTnT, and an automatic tubular chemiluminescence quantitative detection assay was further developed, which presents a high coincidence rate with Roche's.

The post-mortem diagnosis of acute myocardial ischemia remains a challenge for both clinical and forensic pathologists. We performed an experimental study (ligation of left anterior descending coronary artery in rats) in order to identify early markers of myocardial ischemia, to further apply to forensic and clinical pathology in cases of sudden cardiac death. Using immunohistochemistry, Western blots, and gene expression analyses, we investigated a number of markers, selected among those which are currently used in emergency departments to diagnose myocardial infarction and those which are under investigation in basic research and autopsy pathology studies on cardiovascular diseases. The study was performed on 44 adult male Lewis rats, assigned to three experimental groups: control, sham-operated, and operated. The durations of ischemia ranged between 5 min and 24 h. The investigated markers were troponins I and T, myoglobin, fibronectin, C5b-9, connexin 43 (dephosphorylated), JunB, cytochrome c, and TUNEL staining. The earliest expressions (≤30 min) were observed for connexin 43, JunB, and cytochrome c, followed by fibronectin (≤1 h), myoglobin (≤1 h), troponins I and T (≤1 h), TUNEL (≤1 h), and C5b-9 (≤2 h). By this investigation, we identified a panel of true early markers of myocardial ischemia and delineated their temporal evolution in expression by employing new technologies for gene expression analysis, in addition to traditional and routine methods (such as histology and immunohistochemistry). Moreover, for the first time in the autopsy pathology field, we identified, by immunohistochemistry, two very early markers of myocardial ischemia: dephosphorylated connexin 43 and JunB.

Regeneration is a key goal in the design of immunosensors. In this study, we report the temperature-regulated interaction of N-isopropylacrylamide (PNIPAAm) functionalised cardiac troponin T (cTnT) with anti-cTnT. Covalently bonded PNIPAAm on an anti-cTnT bioelectrode showed on/off-switchability, regeneration capacity and temperature triggered sensitivity for cTnT. Above the lower critical solution temperature (LCST), PNIPAAm provides a liphophilic microenvironment with specific volume reduction at the bioelectrode surface, making available binding space for cTnT, and facilitating analyte recognition. Computational studies provide details about the structural changes occurring at the electrode above and below the LCST. Furthermore, free energies associated with the binding of cTnT with PNIPAAm at 25 (ΔGcoil=-6.0 Kcal/mole) and 37 °C (ΔGglobular=-41.0 kcal/mole) were calculated to elucidate the interaction and stability of the antigen-antibody complex. The responsiveness of such assemblies opens the way for miniaturised, smart immuno-technologies with 'built-in' programmable interactions of antigen-antibody upon receiving stimuli., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: To study the skeletal troponin I (skTnI) cross-reactivity of four different commercially available antibodies in four cardiac troponin I (cTnI) research assay versions having the same epitope specificity as evidenced by peptide mapping., Design and Methods: The four research assays all use two solid phase antibodies and one detection antibody attached to intrinsically fluorescent nanoparticles. Two alternative antibodies were used for one capture antibody and two for the detector antibody. The assays were evaluated in terms of analytical sensitivity and by determining assay cross-reactivity to skTnI. Additionally, regression analysis was performed by measuring a sample panel (n=101) with all of the four assay versions., Results: A false-positive cTnI concentration of >7000ng/L was measured with one of the assay versions, when serum was spiked with 500,000ng/L skTnI. The corresponding observed cTnI values for the other three assay versions varied from 616ng/L to 727ng/L. Out of the 101 clinical samples assayed, five showed spuriously (3- to 148-fold) elevated cTnI values with the skTnI interference prone assay setup, but not with the other assay versions. According to our investigational skTnI assay, all five samples contained measurable amounts of skTnI (range: 5500-702,000ng/L)., Conclusions: Two out of four cTnI antibodies tested cross-reacted vastly with skTnI but did not cause any notable interference unless paired together. Therefore, skTnI cross-reactivity should be carefully assessed when cTnI assay antibodies claimed to be cTnI specific are selected., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aim: The role that plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) and cardiac troponins T (cTnT) and I (cTnI) play in supplementing imaging to screen for cardiac late effects remains controversial and the impact of high-sensitivity cTnT and troponin-specific autoantibodies (cTnAAbs) remains unexplored. We studied the role of cardiac biomarkers as indicators of the late effects of anthracyclines among childhood cancer survivors., Methods: We measured NT-proBNP, cTnT, high-sensitivity cTnT, cTnI and cTnAAbs in 76 childhood cancer survivors at a median of 9 years after primary diagnosis. The survivors underwent conventional and real-time three-dimensional echocardiography and 62 underwent cardiac magnetic resonance imaging (MRI)., Results: Of the survivors, four (5.3%) without risk factors for cardiotoxicity were cTnAAb-positive with an impaired cardiac function in MRI. Another four (5.3%) had an abnormal NT-proBNP level associated with an abnormal cardiac function and risk factors for cardiotoxicity. None showed measurable cardiac troponins, determined by the three different methods, with even the high-sensitivity cTnT-levels remaining normal., Conclusion: Elevated plasma NT-proBNP or cTnAAbs indicated that childhood cancer survivors benefitted from being evaluated with modern imaging, despite normal function in conventional echocardiography. However, troponins did not seem to provide additional information on the late cardiotoxicity of anthracyclines., (©2014 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)

Objective: To clone cDNA encoding troponin T of Schistosoma japonicum (SjTnT), and evaluate the protective efficacy induced by recombinant SjTnT in BALB/c mice against S. japonicum challenge infection., Methods: The SjTnT gene was amplified from 28-day-schistosome cDNAs by PCR and then subcloned into pET28a(+). The recombinant SjTnT protein (rSjTnT) was expressed in Escherichia coli BL21 (DE3) cells. The serum specific to rSjTnT was prepared by immunized BALB/c mice with the recombinant antigen, and the immunogenicity of rSjTnT was detected by Western blotting and ELISA. The immuno-protective efficacy induced by rSjTnT in BALB/c mice was evaluated according to the reduction in worm and egg counts., Results: The cDNA encoding SjTnT was successfully cloned and expressed in E. coli. Western blotting showed that rSjTnT had a good immunogenicity. The high level of specific IgG antibodies was detected, and 33.89% worm reduction and 43.94% liver egg reduction were obtained in mice vaccinated with rSjTnT combined with Seppic 206 adjuvant compared with those in the adjuvant control group., Conclusions: rSjTnT could induce partial immuno-protection against S. japonicum infection in BALB/c mice. This study provided a basic for understanding the biological function of SjTnT.

This study demonstrates the development of nanotextured zinc oxide (ZnO) thin films sputter deposited on printed circuit boards (PCB) to enhance the capability in detecting low concentrations of the protein troponin-T. The presence of this particular biomarker in the bloodstream is a direct indicator of current and/or future risk of various forms of cardiovascular diseases. Electrical transduction through impedance spectroscopy was used to detect troponin-T functionalized immunoassays on nanotextured ZnO surfaces. Calibration of the immunoassay was performed by measuring the impedance changes resulting from the binding of increasing concentrations of troponin-T to the immobilized antibodies on the ZnO surface in (i) phosphate buffered saline (PBS) and (ii) human serum. The limit of detection achieved using this platform was 10 fg/mL and 100 fg/mL in PBS and human serum, respectively. Enhanced detection of troponin-T was found to correlate to the oxygen vacancies in the ZnO thin film. PCB was chosen as the substrate for ease of integration with microelectronic device manufacturing., (© 2013 Elsevier B.V. All rights reserved.)

We report lithium ion intercalation mediated efficient exfoliation of graphite to form monolithic graphene sheets which have subsequently been investigated for the development of highly sensitive label-free electrochemical detection platform for cardiac biomarker, Troponin I (cTnI). The spectroscopic and morphological analysis demonstrated the formation of defect free graphene sheets which were successfully employed to fabricate an inter-digited microdevice in a drain-source configuration on a silicon biochip. The graphene gated biochip functionalized with anti-cTnI antibodies used in label free detection of cTnI which exhibited an excellent sensitivity in the picogram range (~1 pg mL(-1)) for cTnI without the use of any enzymatic amplification that promises its potential applicability for bio-molecular detection in clinical diagnosis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: To evaluate the potential involvement of anti-Trypanosoma cruzi and cardiac protein antibody (IgG total and isotypes) production and their possible association with different clinical forms of human chronic Chagas disease., Methods: IgG total and isotypes were measured by ELISA, using epimastigote and trypomastigote forms of T. cruzi as antigens and human cardiac proteins (myosin and troponin T) in sera of patients with indeterminate (IND, n = 72), cardiac (CARD, n = 47) and digestive/cardiodigestive (DIG/CARD-DIG, n = 12) clinical forms of the disease. Samples from uninfected health individuals (CONT, n = 30) and patients with ischaemic cardiomyopathy (ISCH, n = 15) were used as controls. Autoantibody levels were correlated with parameters of cardiac function obtained by electrocardiographic, radiographic and echocardiographic examinations., Results: Fifty five per cent of patients were classified as IND, 35.9% as CARD and 9.1% as DIG/CARD-DIG. Greater total IgG production was observed in IND, CARD and DIG/CARD-DIG chagasic patients than in CONT and ISCH, using trypomastigote, epimastigote and cardiac antigens. Moreover, patients with CARD and DIG/CARD-DIG presented greater total IgG production (trypomastigote and epimastigote antigen) than IND, and a negative correlation was determined between total IgG and left ventricular ejection fraction (LVEF). Patients with IND and CARD presented similar higher levels of total IgG specific to troponin T and myosin than CONT and ISCH individuals. Patients with chronic Chagas disease presented a negative correlation between left ventricular ejection fraction (LVEF) and the production of anti-myosin and troponin T autoantibodies. When grouped as low and high antibody producers and compared with LVEF, we observed that high anti-troponin T (P = 0.042) and myosin (P = 0.013) producers presented lower LVEF than low producers. Moreover, there was a positive correlation (r = 0.9508, P = 0.0001) between the production of troponin T and myosin autoantibodies., Conclusion: These findings indicate that increased production of anti-cardiac troponin T and myosin autoantibodies probably influences the left ventricular ejection fraction and could be related to chagasic cardiomyopathy., (© 2013 John Wiley & Sons Ltd.)

Ventricular hypertrabeculation/noncompaction is a morphologic and functional anomaly of myocardium characterized by prominent trabeculae accompanied by deep recessus. Dilated cardiomyopathy with left ventricular failure is observed in these patients, while the cause or pathophysiologic nature of this complication is not known. Anti-troponin antibodies are formed against circulating cardiac troponins after an acute coronary event or conditions associated with chronic myocyte necrosis, such as dilated cardiomyopathy. In present study, we aimed to investigate cardiac troponins and anti troponin autoantibodies in ventricular noncompaction/hypertrabeculation patients with/without reduced ejection fraction. A total of 50 patients with ventricular noncompaction and 23 healthy volunteers were included in this study. Noncompaction/hypertrabeculation was diagnosed with two-dimensional echocardiography using appropriate criteria. Depending on ejection fraction, patients were grouped into noncompaction with preserved EF (LVEF >50%, n = 24) and noncompaction with reduced EF (LVEF <35%, n = 26) groups. Troponin I, troponin T, anti-troponin I IgM and anti-troponin T IgM were measured with sandwich immunoassay method using a commercially available kit. Patients with noncompaction had significantly higher troponin I (28.98±9.21 ng/ml in NCNE group and 28.11±10.42 ng/ml in NCLE group), troponin T (22.17±6.97 pg/ml in NCNE group and 22.78±7.76 pg/ml in NCLE group) and antitroponin I IgM (1.92±0.43 µg/ml in NCNE group and 1.79±0.36 µg/ml in NCLE group) levels compared to control group, while antitroponin T IgM and IgG were only elevated in patients with noncompaction and reduced EF (15.81±6.52 µg/ml for IgM and 16.46±6.25 µg/ml for IgG). Elevated cardiac troponins and anti-troponin I autoantibodies were observed in patients with noncompaction preceding the decline in systolic function and could indicate ongoing myocardial damage in these patients.

Human cardiac troponin I (hcTnI) and troponin T (hcTnT) are the biomarkers of choice for the diagnosis of cardiac diseases. In an effort to improve assay sensitivity, in this study we developed a novel approach to simultaneously detect hcTnI and hcTnT in homogenous solutions by monitoring enhanced-fluorescence-anisotropy changes. Specifically, our design was based on a competition assay by measuring anisotropy change of fluorophore-labeled peptides bound to primary monoclonal antibodies in the presence of nano-gold-modified secondary antibody in response to the presence of target proteins. Enhanced-fluorescence-anisotropy resulted from interaction between the primary antibody and the nano-gold-labeled secondary antibody, which significantly increased the size and decreased tumbling motion of the complex of peptide-antibodies. The measurements were performed to detect hcTnI and hcTnT either individually or simultaneously in a homogenous buffer solution and in the solutions containing human plasma. Our results showed that when fluorescence emission was monitored at a single wavelength selected by a monochromator the assay at all experimental conditions had excellent linear response to the target proteins within the concentration range of 0.5-40 nM. The detection limit is 0.5 nM for both hcTnI and hcTnT in the presence of human plasma. However, when fluorescence emission was monitored using a cutoff filter, the linear response of the assay to the target proteins is within 15-500 pM. The detection limit is 15 pM which is close to the recommended 99th percentile cutoff point for concentrations of hcTnI and hcTnT tests to discriminate healthy and diseased conditions. Homogenous nature, rapid response time, and easy implementation of our assay design make it a useful tool for disease biomarker and protein sensing.

A novel artificial antibody for troponin T (TnT) was synthesized by molecular imprint (MI) on the surface of multiwalled carbon nanotubes (MWCNT). This was done by attaching TnT to the MWCNT surface, and filling the vacant spaces by polymerizing under mild conditions acrylamide (monomer) in N,N'-methylenebisacrylamide (cross-linker) and ammonium persulphate (initiator). After removing the template, the obtained biomaterial was able to rebind TnT and discriminate it among other interfering species. Stereochemical recognition of TnT was confirmed by the non-rebinding ability displayed by non-imprinted (NI) materials, obtained by imprinting without a template. SEM and FTIR analysis confirmed the surface modification of the MWCNT. The ability of this biomaterial to rebind TnT was confirmed by including it as electroactive compound in a PVC/plasticizer mixture coating a wire of silver, gold or titanium. Anionic slopes of 50 mV decade(-1) were obtained for the gold wire coated with MI-based membranes dipped in HEPES buffer of pH 7. The limit of detection was 0.16 μg mL(-1). Neither the NI-MWCNT nor the MWCNT showed the ability to recognize the template. Good selectivity was observed against creatinine, sucrose, fructose, myoglobin, sodium glutamate, thiamine and urea. The sensor was tested successfully on serum samples. It is expected that this work opens new horizons on the design of new artificial antibodies for complex protein structures., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Designing a surface recognition layer with high anti-fouling ability, high affinity, and high specificity is an important issue to produce high sensitivity biosensing transducers. In this study, a self-assembled monolayer (SAM) consisting of a homogeneous mixture of oligo(ethylene glycol) (OEG)-terminated alkanethiolate and mercaptohexadecanoic acid (MHDA) on Au was employed for immobilizing troponin T antibody and applied in detecting cardiac troponin T by using surface plasmon resonance (SPR). The mixed SAM showed no phase segregation and exhibited human serum albumin resistance, particularly with an antibody-immobilized surface. X-ray photoemission spectra revealed that the chemical composition ratio of OEG to the mixed SAM was 69% and the OEG packing density was 82%. The specific binding of troponin T on the designed surface indicated a good linear correlation (R=0.991, P<0.0009) at concentrations lower than 50 μgmL(-1) with the limit of detection of 100 ngmL(-1) using a SPR measuring instrument. It is concluded that the mixed SAM functions as designed since it has high detection capability, high accuracy and reproducibility, as well as shows strong potential to be applied in rapid clinical diagnosis for label-free detection within 2 min., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: High-sensitivity cardiac troponin assays are being introduced clinically for earlier diagnosis of acute myocardial infarction (AMI). We evaluated the analytical performance of a high-sensitivity cardiac troponin T assay (hscTnT, Roche Diagnostics) in a multicenter, international trial., Methods: Three US and 5 European sites evaluated hscTnT on the Modular® Analytics E170, cobas® 6000, Elecsys 2010, and cobas® e 411. Precision, accuracy, reportable range, an inter-laboratory comparison trial, and the 99th percentile of a reference population were assessed., Results: Total imprecision (CVs) were 4.6-36.8% between 3.4 and 10.3 ng/L hscTnT. Assay linearity was up to 10,000 ng/L and the limit of blank and detection were 3 and 5 ng/L, respectively. The 99th percentile reference limit was 14.2 ng/L (n=533). No significant differences between specimen types, assay incubation time, or reagent lots existed. A substantial positive bias (76%) exists between the 4th generation and hscTnT assays at the low end of the measuring range (<50 ng/L). hscTnT serum pool concentrations were within 2SD limits of the mean of means in the comparison trial, indicating comparable results across multiple platforms and laboratories., Conclusion: The Roche hscTnT assay conforms to guideline precision requirements and will likely identify additional patients with myocardial injury suspicious for AMI., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aims: Chronic heart failure following myocardial infarction (MI) is characterized by progressive left ventricular remodelling (LVR). Despite significant improvements in MI management, LVR remains a frequent complication. Although several risk factors have been identified, such as infarct size, LVR is difficult to predict in clinical practice., Methods and Results: Using a rat model of MI and phosphoproteomic technology, we discovered that remodelling is associated with decreased levels of myocardial and plasma serine(208)-phosphorylated troponin T (TnT). To confirm the association in human plasma, we developed new specific polyclonal antibodies against human/rat serine(207/208)-phosphorylated TnT and tested plasma obtained in the first week after MI from patients with low, intermediate, and high remodelling a year later. We observed a significant decrease of serine(207)-phosphorylated TnT and of the serine(207)-phosphorylated TnT/total TnT ratio in those with intermediate or high LVR. These differences remained statistically significant when adjusted for other determinants of LVR. In contrast, baseline B-type natriuretic peptide levels were not associated with LVR., Conclusion: The level of circulating phosphorylated TnT could be a new biomarker of LVR.

A piezoelectric immunosensor based on gold nanoparticles (AuNPs) co-immobilized on a dithiol-modified surface is proposed for detection of human cardiac troponin T (TnT). Anti-human troponin T (anti-TnT) antibodies were covalently immobilized on the nanostructured electrode surface by thiol-aldehyde linkages. In a homogeneous bulk solution, TnT was captured by anti-TnT immobilized on the QCM electrode. Cyclic voltammetry studies were used to characterize the AuNPs layer on the electrode surface and the anti-TnT immobilization steps. The QCM-flow immunosensor exhibited good reliability, measuring concentrations of TnT from 0.003 to 0.5 ng mL(-1) in human serum with high linearity (r = 0.989; p < 0.01). The immunosensor exhibited a 7% coefficient of variation and 0.0015 ng mL(-1) limit of detection, indicating a high reproducibility and sensitivity. The proposed QCM nanostructured immunosensor is easy to use and has promising potential in the diagnosis of acute myocardial infarction due to its speed and high sensitivity.

Screen-printed electrodes (SPE) have been widely used in the design of disposable sensors bringing advances in the use of electrochemical immunosensors for in field-clinical analysis. In this work, streptavidin polystyrene microspheres were incorporated to the electrode surface of SPEs in order to increase the analytical response of the cardiac troponin T (cTnT), a specific biomarker for the acute myocardial infarction diagnosis. The precise calculation of the stoichiometric streptavidin-biotin ratio [1:4] allowed the increase of sensitivity and stability of the immunosensor response to the cTnT analyte. The surface of the immunosensor was characterized by scanning electron microscopy and cyclic voltammetry. It was observed that the use of streptavidin microspheres significantly increased the analytical sensitivity of the electrode in 8.5 times, showing a curve with a linear response range between 0.1 and 10 ngmL(-1) of cTnT and a detection limit of 0.2 ngmL(-1). The proposed SPE showed ease preparation and high sensitivity allowing the detection of cTnT in the range of clinical levels. The new device coupled with a portable electrochemical analyzer shows great promise for point-of-care quantitative testing of necrosis cardiac proteins., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Troponin T (TnT) is a useful biomarker for studying drug-induced toxicity effects on cardiac cells. We describe how a surface plasmon resonance (SPR) biosensor was applied to monitor the release of TnT from active HL-1 cardiomyocytes in vitro when exposed to cardiotoxic substances. Two monoclonal human TnT antibodies were compared in the SPR immunosensor to analyse the TnT release. The detection limit of TnT was determined to be 30 ng/ml in a direct assay set-up and to be 10 ng/ml in a sandwich assay format. Exposure of the cardiomyocytes to doxorubicin, troglitazone, quinidine and cobalt chloride for periods of 6 and 24 h gave significant SPR responses, whereas substances with low toxicity showed insignificant effects (ascorbic acid, methotrexate). The SPR results were verified with a validated immunochemiluminescence method which showed a correlation of r (2) = 0.790.

Troponin T (TnT) binds to tropomyosin (Tm) to anchor the troponin complex in the thin filament, and it thus serves as a vital link in the Ca(2+) regulation of striated muscle contraction. Pioneer work three decades ago determined that the T1 and T2 chymotryptic fragments of TnT each contains a Tm-binding site. A more precise localization of the two Tm-binding sites of TnT remains to be determined. In the present study, we tested serial deletion constructs of TnT and carried out monoclonal antibody competition experiments to show that the T1 region Tm-binding site involves mainly a 39 amino acids segment in the N-terminal portion of the conserved middle region of TnT. We further employed another set of TnT fragments to locate the T2 region Tm-binding site to a segment of 25 amino acids near the beginning of the T2 fragment. The localization of the two Tm-binding sites of TnT provided new information for the structure-function relationship of TnT and the anchoring of troponin complex on muscle thin filament., (2010 Elsevier Inc. All rights reserved.)

The increased analytical sensitivity of the new generation of methods for cardiac troponin I (cTnI) and T (cTnT) has demonstrated that measurable troponin is present in the blood of healthy adult subjects. These data are not in accordance with the prevailing opinion that any reliably detected increase in cardiac troponins should be considered abnormal and potentially caused by cardiac necrosis. The goal of the present review is to discuss the hypothesis that cardiac troponins can be released from cardiomyocytes, even in healthy adult subjects as a result of a process related to "physiological renewal" of the human myocardium and possibly enhanced by physical exercise or aging. The latest generation of high-sensitive cTnI and cTnT immunoassays are characterized by detection limits (DLs) as low as a few picograms. This clearly represents a greater increase in discrimination than that obtained by the most sophisticated cardiac imaging techniques that are commercially available at present. However, the critical question is whether high-sensitive troponin assays are clinically useful and in particular, whether some specific laboratory biomarkers (such as cTnI and cTnT) yield better diagnostic (or prognostic) accuracy and cost-effectiveness when compared with echocardiography in patients with cardiovascular disease. Only specific and well-designed clinical trials will answer this important question.

Background: Ginsenoside Rb1, a major bioactive component of Panax ginseng, bears various beneficial effects on the cardiovascular system. This study investigated whether ginsenoside Rb1 preconditioning has protective effects on myocardial ischemia-reperfusion injury and its potential mechanism., Methods: Rats subjected to 45 min of myocardial ischemia followed by 120 min of reperfusion were assigned to the following groups: sham-operated, ischemia-reperfusion (I/R), ginsenoside Rb1+I/R, wortmannin(a specific PI3K inhibitor)+I/R, wortmannin drug vehicle (dimethyl sulfoxide, DMSO), wortmannin+sham, ginsenoside Rb1+ wortmannin +I/R. Infarct size was assessed by triphenyltetrazolium chloride staining. Plasma creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB), lactate dehydrogenase (LDH), and troponin T levels were also measured. Akt phosphorylation expression was assessed by immunoblotting., Results: Ginsenoside Rb1 preconditioning reduced infarct size compared with that in the I/R group: 30 +/- 2.6% versus 51 +/- 2.7% (p < 0.01). Ginsenoside Rb1 preconditioning also markedly reduced the plasma CK, CK-MB, LDH and troponin T levels in blood. Akt phosphorylation expression increased after ginsenoside Rb1 preconditioning. These effects of ginsenoside Rb1 preconditioning were significantly inhibited by wortmannin., Conclusion: This is the first study to demonstrate that ginsenoside Rb1 preconditioning has protective effects on myocardial ischemia and reperfusion injury, partly by mediating the activation of the PI3K pathway and phosphorylation of Akt.

The American cockroach, Periplaneta americana, is the predominant cockroach (CR) species in Thailand and a major source of indoor allergens second only to the house dust mite. The incidence of CR allergy among allergic Thai patients is increasing but basic information on the allergenic components is scarce. In this study a recombinant troponin-T was produced by using cDNA prepared from RNA of the P. americana as a template and PCR primers designed from the P. americana troponin-T sequence deposited in the GenBank database. The recombinant protein (Mr approximately 50) did not bind to IgE in the sera of 18 skin prick test positive CR allergic patients. Rabbit polyclonal antiserum (PAb) against the recombinant troponin-T was produced and used in preparing an affinity column for the purification of native troponin-T from the crude P. americana extract (Mr approximately 47). IgE-immunoblotting revealed that the native protein bound to IgE in 3 of the 18 (16.7%) patients. Our results imply that native P. americana troponin-T, but not its recombinant counterpart, is a minor allergen among the CR allergic Thais.

The aim of this study is to investigate the function of the C-terminal extension of three troponin I isoforms, that are unique to the body wall muscles of Caenorhabditis elegans and to understand the molecular interactions within the TN complex between troponin I with troponin C/T, and tropomyosin. We constructed several expression vectors to generate recombinant proteins of three body wall and one pharyngeal troponin I isoforms in Escherichia coli. Protein overlay assays and Western blot analyses were performed using antibodies. We demonstrated that pharyngeal TNI-4 interacted with only the pharyngeal isoforms of troponin C/T and tropomyosin. In contrast, the body wall TNI-2 bound both the body wall and pharyngeal isoforms of these components. Similar to other invertebrates, the N-terminus of troponin I contributes to interactions with troponin C. Full-length troponin I was essential for interactions with tropomyosin isoforms. Deletion of the C-terminal extension had no direct effect on the binding of the body wall troponin I to other muscle thin filament troponin C/T and tropomyosin isoforms.

Mass spectrometric approaches have recently gained increasing access to molecular immunology and several methods have been developed that enable detailed chemical structure identification of antigen-antibody interactions. Selective proteolytic digestion and MS-peptide mapping (epitope excision) has been successfully employed for epitope identification of protein antigens. In addition, "affinity proteomics" using partial epitope excision has been developed as an approach with unprecedented selectivity for direct protein identification from biological material. The potential of these methods is illustrated by the elucidation of a beta-amyloid plaque-specific epitope recognized by therapeutic antibodies from transgenic mouse models of Alzheimer's disease. Using an immobilized antigen and antibody-proteolytic digestion and analysis by high resolution Fourier transform ion cyclotron resonance mass spectrometry has lead to a new approach for the identification of antibody paratope structures (paratope-excision; "parex-prot"). In this method, high resolution MS-peptide data at the low ppm level are required for direct identification of paratopes using protein databases. Mass spectrometric epitope mapping and determination of "molecular antibody-recognition signatures" offer high potential, especially for the development of new molecular diagnostics and the evaluation of new vaccine lead structures.

Background: Cardiac troponins in blood are the most preferred markers of myocardial damage. The fact that they are normally not found in the circulation provides a high level of clinical sensitivity and specificity even when cardiac lesions are small. After myocardial injury, the troponins enter the circulation, where they can be used for diagnosis of acute coronary syndromes. Thus, the cardiac troponins are paramount for disease classification and risk stratification. However, little is known about the long-term effects of the released troponins on cardiac function., Methods and Results: In this study we prepared recombinant murine cardiac troponin I (mc-TnI) and murine cardiac troponin T and used them to immunize mice. We report that A/J mice immunized with mc-TnI developed severe inflammation of the myocardium with increased expression of inflammatory chemokines RANTES (regulated on activation normal T cell expressed and secreted), monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, T-cell activation gene 3, and eotaxin and chemokine receptors CCR1, CCR2, and CCR5. The inflammation was followed by cardiomegaly, fibrosis, reduced fractional shortening, and 30% mortality over 270 days. In contrast, mice immunized with murine cardiac troponin T or with the control buffer showed little or no inflammation and no death. Furthermore, we demonstrate that mice preimmunized with mc-TnI before left anterior descending coronary artery ligation showed greater infarct size, more fibrosis, higher inflammation score, and reduced fractional shortening., Conclusions: Overall, our results show for the first time that provocation of an autoimmune response to mc-TnI induces severe inflammation in the myocardium followed by fibrosis and heart failure with increased mortality in mice.

Background: The cardiac troponins have been shown to be sensitive and specific biochemical markers of myocardial infarction and highly prognostic for future adverse events in patients with acute coronary syndromes. There have been reports suggesting that haemolysis causes a negative interference in the cardiac troponin T (cTnT) assay but the mechanism(s) involved remain unknown. Here we show the effects of haemolysis and haemoglobin per se on the cTnT assay., Methods: The effect of haemolysis was studied by the addition of prepared haemolysate to serum samples with known and clinically relevant cTnT levels. The effect of haemoglobin was studied by the addition of haemoglobin of increasing concentrations and noting its effect on the level of cTnT measured. The effect of putative proteases was determined indirectly by incubating samples with spiked cTnT with various protease inhibitors and observing the changes in the measured cTnT levels., Results: The results show that both haemolysis, which is the release of haemoglobin and corpuscular contents, and haemoglobin itself negatively interfere in the cTnT assay in a concentration-dependent manner, although the former had a greater magnitude of effect. On haemolysis, indirect evidence suggests that proteases are released which degrade the cTnT in serum, thus causing the decreased levels detected. Pepstatin A, a reversible inhibitor of aspartic proteinases, effectively inhibited the loss of cTnT in serum at 37 degrees C and pH 7.4 over a 48-h period. We found that at a haemoglobin level of 0.75 g/L, cTnT declined by more than 10% of the initial concentration, suggesting that falsely decreased levels due to haemolysis may significantly affect the clinical utility of the assay., Conclusions: Haemolysis, haemoglobin per se and possibly proteolysis play a role in the negative interference in cTnT assays. Measures to reduce this interference must be implemented.

We describe here a new approach for the identification of affinity-bound proteins by proteolytic generation and mass spectrometric analysis of their antibody bound epitope peptides (epitope excision). The cardiac muscle protein troponin T was chosen as a protein antigen because of its diagnostic importance in myocardial infarct, and its previously characterised epitope structure. Two monoclonal antibodies (IgG1-1B10 and IgG1-11.7) raised against intact human troponin T were found to be completely cross reactive with bovine heart troponin T. A combination of immuno-affinity isolation, partial proteolytic degradation (epitope excision), mass spectrometric peptide mapping, and database analysis was used for the direct identification of Tn T from bovine heart cell lysate. Selective binding of the protein was achieved by addition of bovine heart cell lysate to the Sepharose-immobilised monoclonal antibodies, followed by removal of supernatant material containing unbound protein. While still bound to the affinity matrix the protein was partially degraded thereby generating a set of affinity-bound, overlapping peptide fragments comprising the epitope. Following dissociation from the antibody the epitope peptides were analysed by matrix assisted laser desorption-ionisation (MALDI) and electrospray-ionisation (ESI) mass spectrometry. The peptide masses identified by mass spectrometry were used to perform an automated database search, combined with a search for a common "epitope motif". This procedure resulted in the unequivocal identification of the protein from biological material with only a minimum number of peptide masses, and requiring only limited mass-determination accuracy. The dramatic increase of selectivity for identification of the protein by combining the antigen-antibody specificity with the redundancy of peptide sequences renders this "affinity-proteomics" approach a powerful tool for mass spectrometric identification of proteins from biological material.

Proteolytic degradation of muscle, which occurs post-mortem as part of the meat-ageing process, results in the production of protein fragments. In beef, degradation of skeletal muscle troponin T (TnT) results in the generation of a 16-residue long peptide (TnT (16-31)), identified in trichloroacetic acid (TCA) soluble extracts. We report the development of a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of TnT (16-31), using polyclonal antibodies raised against synthetic TnT (16-31). The ELISA procedure is based on inhibition of binding of the antibodies to immobilised TnT (16-31) by TnT (16-31) present in solution. Its useful range is 30 pmol to 2 nmol TnT (16-31)/ml. Quantification of TnT (16-31) in TCA muscle extracts showed that its concentration was enhanced with ageing. Moreover, a correlation between TnT (16-31) levels and meat tenderness was observed. The ELISA developed herein may prove advantageous for future use at the research and industrial level.

We examined the immunolocalization of isoforms of muscle proteins, myosin and troponin, in the cremaster muscle of the undescended testis (cryptorchidism). In cryptorchid rats induced by a nonsteroidal androgen antagonist, flutamide, the cremaster muscle contained embryonic myosin and embryonic/cardiac troponin T in both immunofluorescence microscopy and Western blotting using antibodies against myosin and troponin T specific for embryonic, cardiac and fast skeletal muscles. However, in muscles other than the cremaster muscle, i. e., the masseter, pectoral and abdominal muscles, embryonic isoforms of these proteins were undetectable by immunohistochemistry with these antibodies, even in the muscles from cryptorchid rats. Our results showing that high levels of embryonic isoforms of muscle proteins were specifically present in the cremaster muscle of cryptorchid rats induced by flutamide suggest that flutamide treatment of pregnant rats might affect genes controlling the development of the lumbar region of the fetus body resulting in the presence of embryonic protein isoforms in the cremaster muscles which are closely associated with undescended testes.

This study investigated the evolution of a transition metal ion-binding cluster ([H--X--X--X--H](n); Tx) in the alternatively spliced NH(2)-terminal variable region of avian pectoral muscle troponin T (TnT). Encoded by avian fast skeletal muscle TnT-specific P exons, Tx-like structures were expressed in the breast muscle TnT's of almost all birds examined. Their presence results in the developmentally up-regulated high molecular weight pectoral muscle TnT. Sequence analysis and metal affinity chromatography revealed that in Galliformes and Craciformes, the Tx structure evolved into multiple H--X--X--X--H pairs with a high-affinity metal-binding capacity. Turkey, chicken, quail, and curassow breast muscle TnT's contain nine, seven, four, and three consecutive or closely located metal-binding sites, respectively, in the NH(2)-terminal region. The metal-binding affinity of the Tx element increased as the number of His pairs increased due to the duplication of P exons and the conversion of other exon sequences. The data show two related components of avian pectoral muscle TnT evolution: a larger, more acidic NH(2)-terminal segment and a cluster of transition metal-binding sites, both of which may have functional significance for their selection value. The evolution of the Tx segment in the NH(2)-terminal variable region of avian pectoral muscle TnT demonstrates a functional divergence on the basis of tolerance to structural drifting.

Troponin T (TnT) plays an allosteric signal transduction role in the actin thin-filament-based Ca(2+)-regulation of striated muscle contraction. Developmentally regulated alternative RNA splicing produces TnT isoforms differing in their NH(2)-terminal structure. Physical property variations of the NH(2)-terminal hypervariable region of TnT may have a role in tuning the Ca(2+)-sensitivity and overall cooperativity of the muscle. We have previously demonstrated that metal ion or monoclonal antibody binding to the NH(2)-terminal region can modulate the epitopic conformation and troponin I and tropomyosin binding affinity of TnT. To further establish the molecular basis of this conformational and functional modulation, we have characterized the NH(2)-terminal variable region-originated secondary conformational effect in TnT using fluorescence spectral analysis. The chicken fast skeletal muscle TnT isoform, TnT8e16, containing a cluster of transition-metal ion binding sites (Tx) in the NH(2)-terminal variable region was used in this study. TnT8e16 was titrated for Cu(II) binding-induced changes in fluorescence intensity and anisotropy of the COOH-domain Trp residues (W234, W236, and W285), which demonstrated considerable environmental sensitivity in TnT denaturation studies. Nonlinear Stern-Volmer plots of Trp quenching indicated a metal ion binding-induced conformational change in TnT. Fluorescence anisotropy changes upon metal ion binding indicated a decrease in the mobility of the Trp residues and an increase in the flexibility of fluorescein-labeled Cys263 in the COOH domain. These data support a model that the alternatively spliced NH(2)-terminal variable region of TnT modulates conformation and flexibility of other domains of the protein.