High-Sensitivity Sandwich ELISA for Plasma NT-proUcn2: Plasma Concentrations and Relationship to Mortality in Heart Failure

Liew, Oi Wah; Yandle, Timothy G.; Chong, Jenny; Ng, Yan Xia; Frampton, Christopher M.; Ng, Tze Pin; Lam, Carolyn S.P.; Richards, A. Mark

doi:10.1373/clinchem.2015.252932

Cited by 21 publications

(20 citation statements)

References 31 publications

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“…68 Validated ELISA analysis showed that the plasma NT-proUCN2 concentration was significantly increased with heart failure. 69 These findings suggest that UCNs are elevated in patients with heart…”

Section: Crhr and Plasma Ucns In Humansmentioning

confidence: 87%

Corticotropin-Releasing Hormone Family and Their Receptors in the Cardiovascular System

Takefuji

Murohara

2019

Circ J

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acid precursor. 6 CRH plays a key role in regulating the basal and stress-induced pituitary-adrenal axis that increases glucocorticoid and androgen secretion. 7 CRH release in response to acute stress is essential for the survival of the organism, but CRH released as a result of exposure to chronic stress may influence emotions and exert negative effects on the homeostasis of the organism's physiological functions. 8 Urocortin 1 (UCN1),-2, and-3 were identified as a 2nd mammalian CRH family of peptides. Vaughan et al identified UCN1 in the Edinger-Westphal nucleus and lateral superior olive regions of the rat brain as a mammalian member of the mammalian CRH family of peptides. 9 The peptide was named UCN1 because of its homology with fish urotensin and CRH. UCN2 (38 amino acids) and UCN3 (38 amino acids) were identified as members of CRH-like peptides by searching human genome databases and cloning from human and mouse cDNA libraries. 10,11 Simultaneously, human stresscopin (N-terminally 2-amino acid extended UCN3) and stresscopin-related peptide (N-terminally 5-amino acid extended UCN2) were identified as members of the CRH family of peptides by Hsu et al. 12 Although CRH mRNA is expressed widely throughout the brain, UCN mRNA expression has restricted expression levels in the mammalian brain. 13 In peripheral tissue, UCN1 mRNA is broadly expressed in multiple organs, including the pituitary, gastrointestinal tract, testis, heart, G-protein coupled receptors (GPCRs) belong to the largest and most diverse superfamily of cell surface receptors. They react to extracellular stimuli and regulate cardiovascular (CV) function through cellular G-protein-mediated signaling. 1 GPCRs are a conserved family of 7 transmembrane receptors that have been targeted for drug therapy. 2 GPCRs are involved in cardiac dysfunction and hypertension, 3 and inhibitors of GPCRs are widely used to treat patients with CV diseases such as heart failure and hypertension. 4 Several studies have investigated various aspects of 2 GPCR families, β-adrenergic receptors and angiotensin II receptors, in CV diseases. There are approximately 800 GPCRs in humans, but the role of most of the GPCRs in CV diseases remains unclear, suggesting that uncharacterized GPCRs have considerable potential in the development of novel therapeutics for CV diseases. This review focuses on corticotropin-releasing hormone receptors (CRHRs) as potential therapeutic GPCRs in CV diseases, as well as their agonists and antagonists. Corticotropin-Releasing Hormone Family CRH, also named corticotropin-releasing factor, is a peptide that was first characterized from the ovine hypothalamus. 5 CRH is a 41-amino acid polypeptide (Figure) generated by the cleavage of the C-terminus of pre-proCRH, a 196-amino

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Section: Crhr and Plasma Ucns In Humansmentioning

confidence: 87%

Corticotropin-Releasing Hormone Family and Their Receptors in the Cardiovascular System

Takefuji

Murohara

2019

Circ J

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“…When we compared offspring exposed to late-gestation diabetes born to dams on control vs. a HF diet, a lysine demethylase enzyme-encoding gene, Kdm6b , was associated with significant H3K27me3 loss, suggesting potential upregulation of this enzyme. Other notable candidate genes with loss of H3K27me3 occupancy are ABCB9 (associated with type 2 diabetes [65]); Sts (associated with HF diet and ob/ob models of obesity and type 2 diabetes [66]); and Ucn2 (associated with heart failure patients [67]). Additionally, there was significant H3K27me3 loss in the promotor region of ATP5G2 which encodes another subunit in the ATP synthase proton pore.…”

Section: Discussionmentioning

confidence: 99%

Prenatal Exposure to a Maternal High-Fat Diet Affects Histone Modification of Cardiometabolic Genes in Newborn Rats

et al. 2017

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Infants born to women with diabetes or obesity are exposed to excess circulating fuels during fetal heart development and are at higher risk of cardiac diseases. We have previously shown that late-gestation diabetes, especially in conjunction with a maternal high-fat (HF) diet, impairs cardiac functions in rat-offspring. This study investigated changes in genome-wide histone modifications in newborn hearts from rat-pups exposed to maternal diabetes and HF-diet. Chromatin-immunoprecipitation-sequencing revealed a differential peak distribution on gene promoters in exposed pups with respect to acetylation of lysines 9 and 14 and to trimethylation of lysines 4 and 27 in histone H3 (all, false discovery rate, FDR < 0.1). In the HF-diet exposed offspring, 54% of the annotated genes showed the gene-activating mark trimethylated lysine 4. Many of these genes (1) are associated with the “metabolic process” in general and particularly with “positive regulation of cholesterol biosynthesis” (FDR = 0.03); (2) overlap with 455 quantitative trait loci for blood pressure, body weight, serum cholesterol (all, FDR < 0.1); and (3) are linked to cardiac disease susceptibility/progression, based on disease ontology analyses and scientific literature. These results indicate that maternal HF-diet changes the cardiac histone signature in offspring suggesting a fuel-mediated epigenetic reprogramming of cardiac tissue in utero.

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“…ELISA was performed as described previously (13). Assay variability was assessed at 2 concentrations by use of 1 commercial plasma sample supplemented with 72 ng/L or 286 ng/L of NT-proUcn2 and measured across the entire 96-well microplate.…”

Section: Elisa For Testing Whole-plate Uniformitymentioning

confidence: 99%

“…We previously reported a chemiluminescencebased sandwich ELISA to measure plasma Nterminal pro-Urocortin 2 (NT-proUcn2) 6 (13). We observed that while mean intraassay CV was below 5%, mean interassay CV was more than 3-fold higher (see Fig.1 in the Data Supplement that accompanies the online version of this article at http://www.jalm.org/content/vol2/issue5).…”

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confidence: 99%

Variability in Microplate Surface Properties and Its Impact on ELISA

Lilyanna

Moriguchi

et al. 2018

The Journal of Applied Laboratory Medicine

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Background: Microplate-based immunoassays are widely used in clinical and research settings to measure a broad range of biomarkers present in complex matrices. Assay variability within and between microplates can give rise to false-negative and false-positive results leading to incorrect conclusions. To date, the contribution of microplates to this variability remains poorly characterized and described. This study provides new insights into variability in immunoassays attributable to surface characteristics of commercial microplates. Methods: Well-to-well assay variation in γ-treated and nontreated 96-well opaque microplates suitable for chemiluminescence assays was determined by use of a validated sandwich ELISA. Microplate surface characteristics were assessed by sessile drop contact angle measurements, scanning electron microscopy, energy dispersive x-ray spectroscopy, and atomic force microscopy. Results: All microplate types tested exhibited vendor-specific assay response profiles; and "rogue" plates with very high intraassay variation and deviant mean assay responses were found. Within-plate, location-dependent bias in assay responses and variability in well contact angle were also observed. We demonstrate substantial differences in well-surface properties with putative effects on protein-coating reproducibility and hence consistency in immunoassay responses. A surface "cleaning" effect on manufacturing residues was attributed to γirradiation, and treated microplates manifest increased polar functionalities, surface roughness, and assay responses. Conclusions: Our data suggest that tighter control of variability in surface roughness, wettability, chemistry, and level of residual contaminants during microplate preparation is warranted to improve consistency of ELISA assay read out. IMPACT STATEMENT In clinical and research settings, microplate-based immunoassays are subject to assay variability within and between microplates that can give rise to false-negative and false-positive results leading to incorrect conclusions. This article clarifies the contribution of microplates to assay variability, which is poorly understood to date. Detailed physicochemical analyses of microplate well surfaces provided new insights into immunoassay variability attributable to microplate surface properties and preparation processes. Our work prompts further investigations to elucidate surface parameters that influence antibody adsorption and biofunctionality, highlighting manufacturing issues that require further review to overcome problems with microplate variability and assay performance.

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High-Sensitivity Sandwich ELISA for Plasma NT-proUcn2: Plasma Concentrations and Relationship to Mortality in Heart Failure

Cited by 21 publications

References 31 publications

Corticotropin-Releasing Hormone Family and Their Receptors in the Cardiovascular System

Corticotropin-Releasing Hormone Family and Their Receptors in the Cardiovascular System

Prenatal Exposure to a Maternal High-Fat Diet Affects Histone Modification of Cardiometabolic Genes in Newborn Rats

Variability in Microplate Surface Properties and Its Impact on ELISA

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