A sensitive sandwich-enzyme immunoassay for human endothelin

doi:10.1016/0022-1759(89)90012-4

Journal of Immunological Methods

Volume 118, Issue 2, 31 March 1989, Pages 245-250

https://doi.org/10.1016/0022-1759(89)90012-4 Get rights and content

Abstract

A sensitive enzyme immunoassay (EIA) for human endothelin(1–21) has been established. The assay is based on a sandwich method that uses two differing capture and detection anti-endothelin antibodies. A monoclonal anti-endothelin antibody AwETN40, which did not react with an endothelin C-terminal heptapeptide, was used as an immobilized antibody. The Fab′ fragment of rabbit antibodies against the endothelin C-terminal heptapeptide was used as an enzyme-labeled detector antibody after being coupled with horseradish peroxidase (HRP). The assay is sensitive enough to detect as little as 0.2 pg/well (80 amol/well) of endothelin. Preliminary investigations indicated that the basal level of immunoreactive endothelin in male plasma (n = 24) extracted with Seppak C-18 cartridges was 1.59 ± 0.32 pg/ml.

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Endothelins including its most abundant isoform, endothelin-1 (ET-1), are peptides acting as vasoconstrictors when binding to ET_A and ET_B receptors, and, in addition to their distinct roles in normal physiology, endothelins have a central role in the pathophysiology of many diseases including cardiovascular and renal diseases. Endothelin-1 (ET-1), the most potent vasoconstrictor in the cardiovascular system, regulates basal vascular tone and glomerular hemodynamics. ET-1 is involved also in vascular and cardiac hypertrophy, inflammation, and in the development and progression of cardiovascular diseases - e.g. essential hypertension, atherosclerosis, coronary artery disease, congestive heart failure, pulmonary arterial hypertension and cerebrovascular disease and renal diseases - e.g. acute renal failure, polycystic kidney disease and chronic kidney disease. Not surprisingly, the ET system has become a target for therapeutic interventions that now include a few already established and some new promising agents. In this narrative review, we summarize physiologic properties of the ET system, focusing especially on ET-1, and its role in the pathophysiology of ET system activated diseases, and discuss the potentials of therapeutic interventions targeting the ET system in cardiovascular and renal diseases. While ET receptor antagonists have already revolutionized the management of idiopathic pulmonary arterial hypertension, so far, this class of drugs have failed as medication for congestive heart failure. Clinical trials continue to explore new applications of endothelin receptor antagonists in treatment-resistant hypertension and chronic kidney disease and have shown some benefits in the latter group by reducing proteinuria; however, they have not been approved yet. We conclude that larger clinical trials are needed to validate the use of ET receptor antagonists in ET system activated diseases.
Multi-targeted protection of acetaminophen-induced hepatotoxicity in mice by tannic acid
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Briefly, after incubating the mixture of 100 μl serum sample and 200 μl Greiss reagent for 5 min at 37 °C, the tubes were centrifuged, and the optical density of the supernatant was read at 550 nm to determine the NO level. The serum levels of ET-1 were subjected to enzyme immunoassay, as described previously [18]. Peptide concentrations of ET-1 in unknown samples were calculated using a standard curve generated by known concentrations of ET-1.
Tannic acid (TA) is the polyphenol that has beneficial health effects against oxidative stress. However, the hepatoprotective effects of TA are still relatively unknown. In the present study, we evaluated the effects of TA on an acetaminophen (APAP)-induced hepatotoxicity model, which was established by administration of 400 mg/kg of APAP. The levels of alanine transferase (ALT), aspartate transferase (AST), dendothelin-1 (ET-1), nitric oxide (NO) and malondialdehyde (MDA) in the APAP-induced hepatotoxicity mice were significantly increased (up to ~ 200%), while their levels were reduced by pretreatment with TA (25 and 50 mg/kg) (P < 0.05). The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the APAP-induced hepatotoxicity mice were significantly reduced (lower to ~ 65%), while their activities were increased by pretreatment with TA (25 and 50 mg/kg) (P < 0.05). In addition, pretreatment with oral TA (25 and 50 mg/kg) for 3 days before the APAP administration dose-dependently ameliorated changes in hepatic histopathology, suppressed overexpression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), c-fos, c-jun, NF-κB (p65) and caspase-3 (all P < 0.05), downregulated bax and upregulated bcl-2, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) (all P < 0.05) in the liver. These results indicate that TA exhibits significant hepatoprotective effects against APAP-induced hepatotoxicity and suggest that the hepatoprotective mechanisms of TA may be related to anti-oxidation, anti-inflammation and anti-apoptosis.
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These immunogens (40 μg/mouse), together with complete or incomplete Freund’s adjuvant, were injected subcutaneously into 8-week-old female Crj:BALB/c mice at 3-week intervals. Four days after the intravenous injection of 200 μg of the immunogen, spleen cells from each immunized mouse were fused with a mouse myeloma cell line, P3-X63Ag8-U1, as described previously (Suzuki et al., 1989). The anti-rat kisspeptin monoclonal antibodies, M#9-1 (IgG2b, κ) and C#123-3 (IgG1, κ) were selected and purified from the ascites using a Protein A-immobilized column (IPA-300, Repligen, MA, USA).
Age-related disappearance of the LH surge is one of major biomarkers of reproductive aging in female rats. Kisspeptin neurons in the hypothalamic anteroventral periventricular nucleus (AVPV) are proposed as the critical regulator of the preovulatory LH surge in response to estrogenic positive feedback. Here we investigated the possible involvement of the AVPV kisspeptin neurons in the disappearance of the LH surge in middle-age rats. Middle-age rats exhibiting persistent estrus (M-PE) did not show an LH surge although neither Kiss1 mRNA nor peptide in the AVPV was differentially expressed when compared to young rats exhibiting normal estrous cycles (YN). M-PE released LH in response to exogenous kisspeptin in a similar dose-dependent manner as YN, suggesting that their GnRH neurons still maintained responsiveness to kisspeptin. To investigate the estrogenic positive feedback effect on kisspeptin neurons in the AVPV, rats were ovariectomized and supplemented with estradiol (OVX + E₂). We performed in situ hybridization and immunohistochemistry for Kiss1 mRNA and cFos, respectively, and found that M-PE exhibited a significantly lower percentage of Kiss1 mRNA positive neurons with cFos immunoreactivity, although the total number of kisspeptin neurons was not different from that in cyclic rats. Furthermore, OVX + E₂ M-PE did not show the surge-like LH release under high estradiol administration while YN did. Thus our current study suggests that the reduced responsiveness of the AVPV kisspeptin neurons to estrogenic positive feedback presumably results in the decrease in kisspeptin secretion from neurons and eventually causes the age-related disappearance of the LH surge in middle age female rats.
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Development and validation of sensitive sandwich ELISAs for two investigational nonapeptide metastin receptor agonists, TAK-448 and TAK-683
2012, Journal of Pharmaceutical and Biomedical Analysis
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The class of each mAb was determined with a mouse isotype kit (BioRad, Hercules, CA, USA). The reactivity between TAK-448, TAK-683, and the analogs of each mAb obtained from IMG-1, IMG-2, IMG-3, and IMG-4 was investigated by performing cEIAs using horseradish peroxidase (HRP)-labeled IMG-1, IMG-2, IMG-3, or IMG-4, as described previously [27,28]. In brief, 50 μL of TAK-448, TAK-683, or their analogs (0.01, 0.1, 1, 10, 100, or 1000 pmol/mL), 50 μL of diluted mAb solution, and 50 μL of buffer C (sodium phosphate buffer [pH 7.0; 20 mM]) containing 1% bovine serum albumin (BSA), NaCl [0.4 M], and EDTA-2Na [2 mM]) were placed in separate wells of an anti-mouse IgG-coated (1.5 μg/100 μL carbonate buffer (pH 9.0; 0.1 M)/well) 96-well plate (Nunc, Naperville, IL, USA) and incubated at 4 °C for 16–24 h.
TAK-448 and TAK-683, investigational agents with potential utility in the treatment of prostate cancer, are potent low molecular weight metastin receptor agonists consisting of nine amino acids. Monoclonal antibodies (mAbs) against these agents were developed to facilitate their evaluation in preclinical studies. Six mAbs were obtained from four immunogens. Three mAbs recognized the C-terminal of TAK-683 and TAK-448, two recognized the N-terminal of TAK-683, and one recognized the N-terminal of TAK-448. Using various combinations of these six mAbs, sandwich ELISAs for TAK-448 and TAK-683 were developed. These assays were highly sensitive, specific, and accurate. The detection limit for TAK-448 and TAK-683 was 3 and 5 pg/mL, respectively, and there was no interference from rat plasma, rat metastin, or analogs of TAK-448/TAK-683. Recovery achieved ≤±10% with intra-/inter-day assay precision coefficient of variation <10%. The assay demonstrated high stability and sample pre-treatment was not required. Each assay detected the dose-dependent concentration of TAK-448 and TAK-683 in blood 24 h after a single intravenous administration of 0.1 and 1 mg/kg doses.
In conclusion, sensitive sandwich ELISAs were developed to detect the small peptides TAK-448 and TAK-683. The novel assays reliably quantified these nonapeptides in rat plasma, and thus will be useful for preclinical studies of these agents. This methodology may be applicable to the development of similar assays for other short peptides.

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A sensitive sandwich-enzyme immunoassay for human endothelin

Abstract

Biochem. Biophys. Res. Commun.

J. Immunol. Methods

FEBS Lett.

Clin. Chim. Acta

The use of N-hydroxy-5-norbornene-2,3-dicarboximide active esters in peptide synthesis

Chem. Pharmacol. Bull.