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Product Summary
The Quantikine Endothelin-1 immunoassay is a 4.5 hour solid phase ELISA designed to measure ET-1 in cell culture supernates, serum, plasma, and urine. It contains synthetic ET-1 and antibodies raised against synthetic ET-1. This immunoassay has been shown to accurately quantitate synthetic and naturally occurring ET-1.
Sample Values
Serum/Plasma/Urine - Samples from apparently healthy volunteers were evaluated for the presence of Endothelin-1 in this assay. No medical histories were available for the donors used in this study.| Sample Type | Mean (pg/mL) | Range (pg/mL) | Standard Deviation (pg/mL) |
| Human serum (n=35) | 1.24 | 0.47-2.00 | 0.35 |
| Human heparin plasma (n=35) | 1.17 | 0.58-1.96 | 0.32 |
| Mouse serum (n=14) | 3.10 | 1.95-4.03 | 0.58 |
| Mouse EDTA plasma (n=15) | 2.82 | 1.96-3.68 | 0.45 |
| Rat serum (n=15) | 1.63 | 1.32-2.07 | 0.20 |
| Rat EDTA (n=15) | 1.59 | 0.53-2.36 | 0.44 |
| Sample Type | Mean of Detectable (pg/mL) | % Detectable | Range (pg/mL) |
| Human EDTA plasma (n=35) | 1.17 | 97 | ND-1.92 |
| Human urine (n=25) | 0.724 | 48 | 0.44 |
Cell Culture Supernates:
Human peripheral blood leukocytes were cultured in DMEM supplemented with 5% fetal bovine serum, 50 μM beta -mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate. The cells were cultured unstimulated or stimulated with 10 μg/mL PHA for 1 and 6 days. Aliquots of the cell culture supernates were removed and assayed for levels of Endothelin-1.
| Condition | Day 1 (pg/mL) | Day 6 (pg/mL) |
| Unstimulated | ND | 1.29 |
| Stimulated | 3.46 | 7.33 |
Tissue from mice or rats were homogenized and seeded into 100 mL of RPMI containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate for the indicated times. Aliquots of the cell culture supernates were removed and assayed for levels of Endothelin-1.
| Sample Type | Observed Levels (pg/mL) |
| Mouse liver (3 days) | 1.05 |
| Mouse lung (1 day) | 5.15 |
| Rat lung (1 Day) | 20.8 |
Recovery
The recovery of Endothelin-1 spiked to levels throughout the range of the assay in various matrices was evaluated.
| Sample Type | Average % Recovery | Range % |
|---|---|---|
| Cell Culture Media (n=4) | 99 | 88-108 |
| Human EDTA Plasma (n=4) | 93 | 87-102 |
| Human Heparin Plasma (n=4) | 93 | 85-106 |
| Human Serum (n=4) | 98 | 86-107 |
| Mouse EDTA Plasma (n=4) | 99 | 102-107 |
| Mouse Serum (n=4) | 94 | 86-104 |
| Rat EDTA Plasma (n=4) | 99 | 86-113 |
| Rat Serum (n=4) | 98 | 88-113 |
| Urine (n=4) | 91 | 85-109 |
Linearity
To assess the linearity of the assay, samples spiked with high concentrations of Endothelin-1 were serially diluted with Calibrator Diluent to produce samples with values within the dynamic range of the assay.
Scientific Data
Assay Procedure
Refer to the product Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.
- Prepare all reagents, standard dilutions, and samples as directed in the product insert.
- Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
- Add 150 µL of Assay Diluent to each well.
- Add 75 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 1 hour on a horizontal orbital microplate shaker.
- Aspirate each well and wash, repeating the process 3 times for a total of 4 washes.
- Add 200 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 3 hours on the shaker.
- Aspirate and wash 4 times.
- Add 200 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes on the benchtop. PROTECT FROM LIGHT.
- Add 50 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.
150 µL Assay Diluent
75 µL Standard, Control, or Sample
200 µL Conjugate
200 µL Substrate Solution
50 µL Stop Solution
Endothelin-1 Quantikine ELISA Kit Summary
Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Assay Length
4.5 hours
Sample Type & Volume Required Per Well
Cell Culture Supernates (75 uL), Serum (75 uL), EDTA Plasma (75 uL), Heparin Plasma (75 uL), Urine (75 uL)
Sensitivity
0.207 pg/mL
Assay Range
0.4 - 25 pg/mL (Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma, Urine)
Specificity
Synthetic and natural Endothelin-1
Cross-reactivity
Cross-reactivity observed with 1 or more available related molecules.< 50% cross-species reactivity observed with species tested.
Interference
No significant interference observed with available related molecules.
背景
别名
ARCND3,EDN1,endothelin 1,Endothelin-1,ET1,HDLCQ7,PPET1,preproendothelin-1,QME
背景
Background: Endothelin-1
Endothelin-1 (ET-1), a peptide of 21 amino acid (aa) residues, is a pleiotropic molecule best known for its action as a potent vasoconstrictor (1). Originally isolated from porcine aortic endothelial cells, ET-1 is one of a family of three proteins encoded by distinct genes that also includes Endothelin-2 (ET-2) and Endothelin-3 (ET-3) (2, 3). ET-2 and ET-3 differ from ET-1 by 2 and 6 amino acids, respectively (1, 2). All members of the Endothelin family contain two essential disulfide bridges and six conserved aa residues at the C-terminus. Human ET-1 is initially synthesized as a pre-pro-polypeptide of 212 amino acids (2, 4). It is proteolytically cleaved by a signal peptidase to produce pro-ET-1 and further processed by a Furin-like protease to yield a 38 aa peptide termed Big ET-1 (5, 6). Big ET-1 is then cleaved by the membrane-bound metalloprotease Endothelin-converting enzyme (ECE-1), producing the potent 21 aa mature form ET-1 (aa 1-21) (7, 8). Alternatively, ET-1 may exist in an active 31 aa form (ET-1 (aa 1-31)) following cleavage of Big ET-1 by chymase (9-12). The vascular endothelium is an abundant source of ET-1 (3, 13). It may also be expressed by leukocytes, smooth muscle cells, mesangial cells, cardiac myocytes, and astrocytes (14, 15). ET-1 can be induced in endothelial cells by many factors including mechanical stimulation, various hormones, and pro-inflammatory cytokines (16). Production is inhibited by nitric oxide (NO), Prostacyclin, and atrial natriuretic peptide (ANP) (17-19).
Two receptors for the Endothelin family have been cloned and designated ETA and ETB (20-23). ETA and ETB belong to the large family of heptahelical G protein-coupled receptors. The ETA receptor shows a higher affinity for ET-1 than for ET-2 and lowest affinity for ET-3, while the ETB receptor shows approximately equal affinity for each of the three Endothelins (21, 22, 24). ETA is primarily responsible for the vasoconstrictor effects of ET-1 and is expressed by blood vessel smooth muscle cells (25, 26). The ETB receptor is also present in smooth muscle and the endothelia of blood vessels, kidney, lung, and brain (27). ET-1 has the ability to activate an array of signaling cascades including classical phosphatidylinositol turnover pathways leading to downstream PKC activation and Ca2+ mobilization (28-32). Other potential signaling mediators activated or produced by ET-1 include PI 3-kinase/Akt, NO, FAK, and Rho GTPases (32-37). ET-1 signaling may also be mediated indirectly via transactivation of the EGF receptor leading to downstream signaling by Ras and MAP kinases (38, 39). Injection of a single dose of ET-1 produces an initial decrease in systemic blood pressure followed by a prolonged increase in blood pressure (16, 40, 41). Blockade of Endothelin receptors with a systemic injection of an ETA/ETB antagonist causes progressive vasodilation, and elevated levels of ET-1 are found in some forms of human hypertension (42, 43). ET-1 also stimulates cardiac contraction and the growth of cardiac myocytes, regulates the release of vasoactive substances, and stimulates smooth muscle cell mitogenesis (32, 44-46). It also acts as a pro-survival factor for endothelial cells and regulates secretion by hypothalamic and pituitary cells (47, 48). ET-1 may control inflammatory responses by promoting the adhesion and migration of neutrophils and stimulating the production of pro-inflammatory cytokines (49-53). It has also been implicated in cancer progression at several levels including regulating the proliferation and migration of tumor cells and acting as a pro-angiogenic factor (54, 55). In addition, ET-1 has putative roles in other pathologies including septic shock, atherosclerosis, heart failure, renal insufficiency, pulmonary hypertension, and cerebrovascular conditions associated with subarachnoid hemorrhage (15, 56-63).
Entrez Gene IDs:
1906 (Human); 13614 (Mouse); 24323 (Rat)
Alternate Names:
ARCND3; EDN1; endothelin 1; Endothelin-1; ET1; HDLCQ7; PPET1; preproendothelin-1; QME
研究领域
制备和贮存
保存方式
Preparation and Storage
Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
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