Development Of High-sensitivity Assays Research Articles

Cardiac biomarker measurement by point of care testing – Development, rationale, current state and future developments

Cardiac biomarker measurements are integral to the diagnosis and management of patients presenting with breathlessness and chest pain. Measurement of B type natriuretic peptide either directly or of the N-terminal portion of the prohormone although possible by point of care testing (POCT) has largely become a laboratory test. Measurement of the cardiac troponins cardiac troponin T (cTnT) and cardiac troponin I (cTnI) can easily and accurately be performed by POCT. The situation has been complicated by the development of high sensitivity assays for cTnT and cTnI and the subsequent development of rapid rule out algorithms allowing patient categorisation and discharge on admission and 1 to 2 h following admission. This article reviews the development of POCT for cardiac biomarkers, the evidence base comparing POCT with central laboratory testing, its strengths and limitations, and how POCT fits into the world of high sensitivity troponin assays. It also discusses what evidence there is that POCT can form part of rapid decision-making strategies and how this applies in an era of algorithms based on and is derived from measurement of high sensitivity troponin in the central laboratory.

A universal multiplex assay format for ultra-sensitive cytokine quantification in human serum and plasma

Abstract Quantifying prospective biomarkers of immune signaling in biological samples requires high assay sensitivity. The Simoa Planar Array provides greater sensitivity than ELISA, but has a finite assay menu. This study aimed to develop a universal multiplex ‘homebrew’ planar immunoassay format for development of high sensitivity assays for any analyte. Anchor antibodies specific for a peptide tag were microprinted in microwell plates. The peptide tags were conjugated to analyte-specific capture antibodies via maleimide chemistry, enabling high affinity binding of capture to anchor antibody. Bound sample antigen was sandwiched between the peptide-tagged capture and biotinylated detector antibodies; chemiluminescent detection was used to quantify signal. This ‘homebrew’ approach successfully detected sub picomolar levels of cytokines and chemokines in human serum and plasma in a multiplex assay measuring IL-5, IL-6, IL-10, IL-22, TNFa. When compared to commercially available planar assays the universal planar assay format provided comparable LLOQ and LOD with sample correlation (R2) >0.97 for all analytes tested. The universal planar assay was used to successfully screen multiple antibody pairs for human and mouse biomarkers and to develop robust assays using mouse, rabbit or goat monoclonal and polyclonal antibodies. This universal multiplex planar homebrew assay format combines the accuracy, reproducibility and high sensitivity of Simoa Planar Array technology with total analyte flexibility, and provides assay developers a simple and versatile method to rapidly develop robust high-sensitivity multiplex assays for any analyte of their choice.

A universal planar assay format for high sensitivity cytokine quantification in human serum and plasma

Abstract Quantifying prospective biomarkers of immune signaling in biological samples requires high assay sensitivity. The Simoa Planar Array provides greater sensitivity than ELISA, but has a finite assay menu. This study aimed to develop a universal ‘homebrew’ planar immunoassay format for development of high sensitivity assays for any analyte, by using an anchor antibody/peptide tag system to develop a universal capture plate. Anchor antibody specific for a peptide tag was microprinted in microwell plates. The peptide tag was conjugated to analyte-specific capture antibodies via maleimide chemistry, enabling high affinity binding of capture to anchor antibody. Bound sample antigen was sandwiched between the peptide-tagged capture and biotinylated detector antibodies; chemiluminescent detection was used to quantify signal. This ‘homebrew’ approach successfully detected sub picomolar levels of cytokines and chemokines in human or mouse serum and plasma, including IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22, ITAC, MIP1a and MIP3b. When compared to commercially available planar assays the universal planar assay format provided comparable LLOQ and LOD with sample correlation (R2) >0.97 for all analytes tested. The universal planar assay was used to successfully screen multiple antibody pairs for human and mouse biomarkers and to develop robust assays using mouse, rabbit or goat monoclonal and polyclonal antibodies. This universal planar homebrew assay format combines the accuracy, reproducibility and high sensitivity of Simoa Planar Array technology with total analyte flexibility, and provides assay developers a simple and versatile method to rapidly develop robust high-sensitivity assays for any analyte of their choice.

Abstract 9326: Peri-Operative Myocardial Damage in Higher-Risk Patients Undergoing Elective Non-Cardiac Surgery

Introduction: The development of high-sensitivity assays has greatly improved the analytical performance of cardiac troponin, allowing detection of very minor damage to cardiac muscle. High sensitivity troponin (hsTnT) is elevated in subgroups of stable populations, with prognostic significance. We postulated hsTnT would be elevated in a proportion of patients undergoing elective non-cardiac surgery, with this elevation predicting adverse peri-operative events. Aims: The aims of our study were to identify the incidence of elevated hsTnT in patients undergoing non-cardiac surgery, and to determine the association between elevated pre-operative hsTnT and adverse peri-operative events. Methods: Consecutive patients aged ≥45 years undergoing major elective non-cardiac surgery, prescribed anti-platelet therapy for primary or secondary cardiovascular event prevention, were included. Baseline, 24 and 48-hour post-operative hsTnT (Roche Diagnostics, 99th percentile 13ng/L) were collected, an elevation above the 99th percentile or if baseline hsTnT elevated, a delta value of 30%, used to define myocardial damage. Results: Included in the analysis were 160 consecutive patients undergoing elective non-cardiac surgery (mean age 72.3 ± 9.7 years, 66% male). Baseline hsTnT was elevated in 29.5% of all patients. Peri-operative myocardial damage occurred in 28.6% of patients. Independent predictors of peri-operative myocardial damage included age (HR 1.07, 1.02-1.12), intra-operative hypotension (HR 3.65, 1.34-9.91), and pre-operative haemoglobin (Hb) concentration (HR 0.97, 0.95-0.99). Baseline elevated hsTnT did not predict peri-operative myocardial damage. Myocardial infarction as defined by the Universal Definition occurred in only 2.7% of patients. Conclusions: Although pre-operative hsTnT was commonly elevated in this cohort, there was no significant association with post-operative myocardial damage. We found 28% of stable higher-risk patients undergoing elective non-cardiac surgery have hsTnT determined peri-operative myocardial damage. The significant associations of intra-operative hypotension and pre-operative Hb concentration suggest myocardial damage is predominantly due to a supply and demand mismatch.

Synthesis of fluorescently labelled rhamnosides: probes for the evaluation of rhamnogalacturonan II biosynthetic enzymes

Three fluorescently labelled saccharides 10–12, representing structures found in pectic glycan rhamnogalacturonan II (RG-II), were synthesised by chemical glycosylation of O-6 of diacetone-d-galactose followed by deprotection and reductive amination with amino-substituted fluorophore APTS. This convenient method installs a common aminogalactitol-based tether in order to preserve the integrity of the reducing end of specific carbohydrates of interest. APTS-labelled glycans prepared in this manner were purified by carbohydrate gel electrophoresis and subjected to capillary electrophoresis analysis, as a basis for the subsequent development of high sensitivity assays for RG-II-active enzymes.

Blood cardiac troponin in toxic myocardial injury: archetype of a translational safety biomarker

A translational safety biomarker for toxic myocardial injury is needed in drug discovery and development. This need reflects the increasing recognition of occurrence of cardiotoxicities, prior lack of preclinical blood biomarkers for toxic cardiac injury, introduction of troponin as a biomarker, and regulatory and industry drivers. Cardiac troponin is considered the gold-standard biomarker in humans for cardiac injury due to ischemic injury and drug toxicity. It has been demonstrated to correlate highly with histopathological extent of injury, degree of impairment of cardiac function, and prognosis. Numerous studies have now clearly demonstrated that both cardiac troponin T and cardiac troponin I are sensitive and specific biomarkers of cardiac injury in laboratory animals. Their use is highly recommended for incorporation into preclinical drug-safety studies, especially whenever there is any history of cardiac effect in prior studies with a compound of the same or similar chemical or pharmacological class. The main caveats with respect to cross-species use of specific cardiac troponin assays are the need for species-specific validation, definition of cut-offs based on relevant assessments of imprecision and reference ranges or concurrent controls, and knowledge of the species-dependent kinetics of release into, and clearance from, the blood. Future development of high-sensitivity assays should determine whether minimal increases below a threshold concentration of troponin might reflect reversible myocardial effects.