Search

Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1-8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57-60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits., (© 2024. The Author(s).)

This chapter describes an epitope-directed approach to generate antipeptide monoclonal antibodies to multiple nonoverlapping protein sites using a cocktail of fusion peptides as immunogen. It provides a step-by-step protocol on how antigenic peptides on a target protein can be identified by in silico prediction and discusses considerations for final peptide selection. Each antigenic peptide (10-20 amino acids long) is displayed as three-copy inserts on the surface exposed loop of a thioredoxin scaffold protein. The corresponding DNA coding sequence specifying the tripeptide insert flanked by Gly-Ser-Gly-Ser-Gly linkers is cloned in-frame into the Rsr II site of the thioredoxin gene in the pET-32a vector. The presence of a C-terminal polyhistidine tag (His 6 -tag) allows the soluble fusion proteins to be purified by one-step native immobilized metal affinity chromatography (IMAC) to greater than 95% purity. Multiple thioredoxin fusion proteins are mixed in equimolar concentrations and used as an immunogen cocktail for animal immunization. The use of short antigenic peptides of known sequence facilitates direct epitope mapping requiring only small mutagenesis scan peptide libraries in the multipin peptide format., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) inactivation is an important step toward enhanced biosafety in testing facilities and affords a reduction in the biocontainment level necessary for handling virus-positive biological specimens. Virus inactivation methods commonly employ heat, detergents, or combinations thereof. In this work, we address the dearth of information on the efficacy of SARS-CoV-2 inactivation procedures in plasma and their downstream impact on immunoassays. We evaluated the effects of heat (56 °C for 30 min), detergent (1-5% Triton X-100), and solvent-detergent (SD) combinations [0.3-1% tri- n -butyl phosphate (TNBP) and 1-2% Triton X-100] on 19 immunoassays across different assay formats. Treatments are deemed immunoassay-compatible when the average and range of percentage recovery (treated concentration relative to untreated concentration) lie between 90-110 and 80-120%, respectively. We show that SD treatment (0.3% TNBP/1% Triton-X100) is compatible with more than half of the downstream immunoassays tested and is effective in reducing SARS-CoV-2 infectivity in plasma to below detectable levels in plaque assays. This facile method offers enhanced safety for laboratory workers handling biological specimens in clinical and research settings., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Background and Objective: Patients with persistent asthma often show poor adherence to inhaled corticosteroids (ICS). Shared decision-making can improve adherence rates in this population. Patient decision aids (PDAs) are tools to facilitate shared decision-making. To date, only one PDA, developed in a Canadian French-speaking population, exists for patients considering ICS maintenance therapy. This PDA has been culturally adapted in this study to contextualize to the needs of multi-ethnic Asian patients in Singapore. This study explored the views of local clinicians on the content, design and implementation of this newly-adapted PDA., Methods: 24 clinicians, who were purposively sampled from polyclinics and a tertiary institution, were interviewed on the content, design and implementation of the PDA. The interviews were audio-recorded, transcribed and analyzed via thematic analysis., Results: Clinicians generally accepted the design of the PDA. They suggested for the target users to be patients on Step 2 of GINA guidelines and the number of options to be reduced from four to two (do nothing or start inhaled corticosteroids). Moreover, they supported including a list of values for patients to select from given that Asian patients often do not articulate their values readily. The addition of more visual aids, the production of multilingual Asian editions and the involvement of nurses to administer the PDA was also suggested., Conclusion: The PDA was culturally-adapted with local clinicians' perspectives to target multi-ethnic Asian patients with persistent asthma (Step 2 GINA guidelines). The main changes include a list of values and addition of visual aids.

Background: Lack of validation and standardization of research-use-only (RUO) immunoassays brings with it inherent threats to authenticity and functional quality. Poor correlation between different commercial neprilysin RUO immunoassays is concerning and discordant findings need to be resolved. We seek to identify and validate reliable neprilysin immunoassays to strengthen the scientific rigor and reproducibility of neprilysin-related investigation and of biomarker research in general., Methods: Soluble neprilysin (sNEP) concentrations were determined in cohorts (n = 532) from Spain (Cohort 1), New Zealand (NZ, Cohort 2) and Singapore (Cohort 3), using commercial kits from six vendors. Apparent sNEP concentrations were correlated between different assays and with plasma neprilysin activity. Assay reliability was further validated by performance verification, MS analysis and cross-reactivity tests., Results: sNEP in Cohorts 1 and 2 measured concurrently in Spain and NZ showed significant inter-laboratory correlation only for the Aviscera Bioscience sNEP ELISA SK00724-01. Neprilysin concentrations obtained with the R&D systems and SK00724-01 ELISAs correlated with each other but not with neprilysin activity. In Cohort 3, sNEP concentrations from the Perkin Elmer AlphaLISA and Biotechne ELLA assays agreed (r = 0.89) and both correlated with neprilysin activity (r = 0.87, 0.77 respectively). MS analysis detected authentic neprilysin in the AlphaLISA kit calibrator and in antibody pull-down material from human plasma. The AlphaLISA assay performed within acceptable limits (spike and recovery, dilutional linearity, inter- and intra-assay CV) and showed no cross-reactivity against neprilysin substrates and closely-related analogues., Conclusion: AlphaLISA and ELLA assays provide reliable measures of sNEP concentrations. Reliability of other commercial neprilysin assays remains in question., (Copyright © 2022. Published by Elsevier Inc.)

Aim: Dispatcher-assisted cardiopulmonary resuscitation (DA-CPR) can increase bystander CPR rates and improve outcomes in out-of-hospital cardiac arrest (OHCA). Despite the use of protocols, dispatchers may falsely recognise some cases to be in cardiac arrest. Hence, this study aimed to find the incidence of DA-CPR initiated for non-OHCA cases, its characteristics and clinical outcomes in the Singapore population., Methods: This was a multi-centre, observational study of all dispatcher-recognised cardiac arrests cases between January to December 2017 involving three tertiary hospitals in Singapore. Data was obtained from the Pan-Asian Resuscitation Outcomes Study cohort. Audio review of dispatch calls from the national emergency ambulance service were conducted and information about patients' clinical outcomes were prospectively collected from health records. Univariate analysis was performed to determine factors associated with in-hospital mortality among non-OHCA patients who received DA-CPR., Results: Of the 821 patients recognised as having OHCA 328 (40.0%) were not in cardiac arrest and 173 (52.7%) of these received DA-CPR. No complications from chest compressions were found from hospital records. The top diagnoses of non-OHCA patients were cerebrovascular accidents (CVA), syncope and infection. Only final diagnoses of CVA (aOR 20.68), infection (aOR 17.34) and myocardial infarction (aOR 32.19) were significantly associated with in-hospital mortality., Conclusion: In this study, chest compressions initiated on patients not in cardiac arrest by dispatchers did not result in any reported complications and was not associated with in-hospital mortality. This provides reassurance for the continued implementation of DA-CPR., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Most randomized trials on minimally invasive cholecystectomy have been conducted with standard (3/4-port) laparoscopic or open cholecystectomy serving as the control group. However, there exists a dearth of head-to-head trials that directly compare different minimally invasive techniques for cholecystectomy (eg, single-incision laparoscopic cholecystectomy versus needlescopic cholecystectomy). Hence, it remains largely unknown how the different minimally invasive cholecystectomy techniques fare up against one another., Methods: To minimize selection and confounding biases, only randomized controlled trials were considered for inclusion. Perioperative outcomes were compared using frequentist network meta-analyses. The interpretation of the results was driven by treatment effects and surface under the cumulative ranking curve values. A sensitivity analysis was also undertaken focusing on a subgroup of randomized controlled trials, which recruited patients with only uncomplicated cholecystitis., Results: Ninety-six eligible randomized controlled trials comprising 11,083 patients were identified. Risk of intra-abdominal infection or abscess, bile duct injury, bile leak, and open conversion did not differ significantly between minimally invasive techniques. Needlescopic cholecystectomy was associated with the lowest rates of wound infection (surface under the cumulative ranking curve value = 0.977) with an odds ratio of 0.095 (95% confidence interval: 0.023-0.39), 0.32 (95% confidence interval: 0.11-0.98), 0.33 (95% confidence interval: 0.11-0.99), 0.36 (95% confidence interval: 0.14-0.98) compared to open cholecystectomy, single-incision laparoscopic cholecystectomy, mini-laparotomy, and standard laparoscopic cholecystectomy, respectively. Mini-laparotomy was associated with the shortest operative time (surface under the cumulative ranking curve value = 0.981) by a mean difference of 22.20 (95% confidence interval: 13.79-30.62), 12.17 (95% confidence interval: 1.80-22.54), 9.07 (95% confidence interval: 1.59-16.54), and 8.36 (95% confidence interval: -1.79 to 18.52) minutes when compared to single-incision laparoscopic cholecystectomy, needlescopic cholecystectomy, standard laparoscopic cholecystectomy, and open cholecystectomy, respectively. Needlescopic cholecystectomy appeared to be associated with the shortest hospitalization (surface under the cumulative ranking curve value = 0.717) and lowest postoperative pain (surface under the cumulative ranking curve value = 0.928)., Conclusion: Perioperative outcomes differed across minimally invasive techniques and, in some instances, afforded superior outcomes compared to standard laparoscopic cholecystectomy. These findings suggest that there may be equipoise for exploring further the utility of novel minimally invasive techniques and potentially incorporating them into the general surgery training curriculum., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Clinicians need improved tools to better identify nonacute heart failure with preserved ejection fraction (HFpEF)., Objectives: The purpose of this study was to derive and validate circulating microRNA signatures for nonacute heart failure (HF)., Methods: Discovery and validation cohorts (N = 1,710), comprised 903 HF and 807 non-HF patients from Singapore and New Zealand (NZ). MicroRNA biomarker panel discovery in a Singapore cohort (n = 546) was independently validated in a second Singapore cohort (Validation 1; n = 448) and a NZ cohort (Validation 2; n = 716)., Results: In discovery, an 8-microRNA panel identified HF with an area under the curve (AUC) 0.96, specificity 0.88, and accuracy 0.89. Corresponding metrics were 0.88, 0.66, and 0.77 in Validation 1, and 0.87, 0.58, and 0.74 in Validation 2. Combining microRNA panels with N-terminal pro-B-type natriuretic peptide (NT-proBNP) clearly improved specificity and accuracy from AUC 0.96, specificity 0.91, and accuracy 0.90 for NT-proBNP alone to corresponding metrics of 0.99, 0.99, and 0.93 in the discovery and 0.97, 0.96, and 0.93 in Validation 1. The 8-microRNA discovery panel distinguished HFpEF from HF with reduced ejection fraction with AUC 0.81, specificity 0.66, and accuracy 0.72. Corresponding metrics were 0.65, 0.41, and 0.56 in Validation 1 and 0.65, 0.41, and 0.62 in Validation 2. For phenotype categorization, combined markers achieved AUC 0.87, specificity 0.75, and accuracy 0.77 in the discovery with corresponding metrics of 0.74, 0.59, and 0.67 in Validation 1 and 0.72, 0.52, and 0.68 in Validation 2, as compared with NT-proBNP alone of AUC 0.71, specificity 0.46, and accuracy 0.62 in the discovery; with corresponding metrics of 0.72, 0.44, and 0.57 in Validation 1 and 0.69, 0.48, and 0.66 in Validation 2. Accordingly, false negative (FN) (81% Singapore and all NZ FN cases were HFpEF) as classified by a guideline-endorsed NT-proBNP ruleout threshold, were correctly reclassified by the 8-microRNA panel in the majority (72% and 88% of FN in Singapore and NZ, respectively) of cases., Conclusions: Multi-microRNA panels in combination with NT-proBNP are highly discriminatory and improved specificity and accuracy in identifying nonacute HF. These findings suggest potential utility in the identification of nonacute HF, where clinical assessment, imaging, and NT-proBNP may not be definitive, especially in HFpEF., (Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)