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Endothelin-1 Quantikine ELISA Kit

R&D Systems | Catalog # DET100

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Key Product Details

Assay Length

4.5 hours

Sample Type & Volume Required Per Well

Cell Culture Supernates (75 µL), Serum (75 µL), EDTA Plasma (75 µL), Heparin Plasma (75 µL), Urine (75 µL)

Sensitivity

0.207 pg/mL

Assay Range

0.39-25 pg/mL (Cell Culture Supernates, Serum, EDTA Plasma, Heparin Plasma, Urine)
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Product Summary for Endothelin-1 Quantikine ELISA Kit

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.

Product Specifications

Assay Type

Solid Phase Sandwich ELISA

Format

96-well strip plate

Measurement

Quantitative ELISA

Detection Method

Colorimetric - 450nm (TMB)

Conjugate

HRP

Species

Multi-Species

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.

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 TypeMean (pg/mL)Range (pg/mL)Standard Deviation (pg/mL)
Human serum (n=35)1.240.47-2.000.35
Human heparin plasma (n=35)1.170.58-1.960.32
Mouse serum (n=14)3.101.95-4.030.58
Mouse EDTA plasma (n=15)2.821.96-3.680.45
Rat serum (n=15)1.631.32-2.070.20
Rat EDTA (n=15)1.590.53-2.360.44

Sample TypeMean of Detectable (pg/mL)% DetectableRange (pg/mL)
Human EDTA plasma (n=35)1.1797ND-1.92
Human urine (n=25)0.724480.44
ND=Non-detectable

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.

ConditionDay 1 (pg/mL)Day 6 (pg/mL)
UnstimulatedND1.29
Stimulated3.467.33
ND=Non-detectable

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 TypeObserved Levels (pg/mL)
Mouse liver (3 days)1.05
Mouse lung (1 day)5.15
Rat lung (1 Day)20.8





Precision

Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.

Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.

Cell Culture Supernates, EDTA Plasma, Heparin Plasma, Serum, Urine

Intra-Assay Precision Inter-Assay Precision
Sample 1 2 3 1 2 3
n 20 20 20 40 40 40
Mean (pg/mL) 3.00 7.34 14.7 3.05 7.43 14.4
Standard Deviation 0.120 0.170 0.280 0.231 0.438 0.759
CV% 4.0 2.3 1.9 7.6 5.9 5.3

Recovery for Endothelin-1 Quantikine ELISA Kit

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.

Multi-species Endothelin-1 ELISA Linearity

Scientific Data Images for Endothelin-1 Quantikine ELISA Kit

Multi-species Endothelin-1 ELISA Standard Curve

Multi-species Endothelin-1 ELISA Standard Curve

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

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).

Alternate Names

ARCND3, EDN1, ET1, HDLCQ7, PPET1, QME

Entrez Gene IDs

1906 (Human); 13614 (Mouse); 24323 (Rat)

Gene Symbol

EDN1

Additional Endothelin-1 Products

Product Documents for Endothelin-1 Quantikine ELISA Kit

Certificate of Analysis

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Product Specific Notices for Endothelin-1 Quantikine ELISA Kit

For research use only

⚠ WARNING: This product can expose you to chemicals including N,N-Dimethylforamide, which is known to the State of California to cause cancer. For more information, go to www.P65Warnings.ca.gov.

Citations for Endothelin-1 Quantikine ELISA Kit

Customer Reviews for Endothelin-1 Quantikine ELISA Kit (9)

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  • Endothelin-1 Quantikine ELISA Kit
    Name: Anonymous
    Sample Tested: rat plasma and rat serum
    Verified Customer | Posted 12/15/2023
    The kit worked excellent for rat plasma and serum.
  • Endothelin-1 Quantikine ELISA Kit
    Name: Anonymous
    Sample Tested: Plasma
    Verified Customer | Posted 11/10/2023
    Endothelin-1 Quantikine ELISA Kit DET100
  • Endothelin-1 Quantikine ELISA Kit
    Name: Anonymous
    Sample Tested: Plasma and Urine
    Verified Customer | Posted 11/16/2022
    Plasma was in range at 4-fold dilution. Urine was in range at 2-fold dilution.
    Endothelin-1 Quantikine ELISA Kit DET100
  • Endothelin-1 Quantikine ELISA Kit
    Name: Anonymous
    Sample Tested: EDTA Plasma
    Verified Customer | Posted 08/22/2019
    Endothelin-1 Quantikine ELISA Kit DET100
  • Endothelin-1 Quantikine ELISA Kit
    Name: Anonymous
    Sample Tested: Urine
    Verified Customer | Posted 12/17/2018
    Endothelin-1 Quantikine ELISA Kit DET100
  • Endothelin-1 Quantikine ELISA Kit
    Name: Christian Cassel
    Sample Tested: Serum and Plasma
    Verified Customer | Posted 10/11/2018
  • Endothelin-1 Quantikine ELISA Kit
    Name: Anonymous
    Sample Tested: Urine
    Verified Customer | Posted 09/11/2018
    Endothelin-1 Quantikine ELISA Kit DET100
  • Endothelin-1 Quantikine ELISA Kit
    Name: Anonymous
    Sample Tested: Plasma
    Verified Customer | Posted 10/23/2017
    Plasma was used from male mice.
    Endothelin-1 Quantikine ELISA Kit DET100
  • Endothelin-1 Quantikine ELISA Kit
    Name: Anonymous
    Sample Tested: Serum and Plasma
    Verified Customer | Posted 08/02/2017
    Endothelin-1 Quantikine ELISA Kit DET100

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Protocols

View specific protocols for Endothelin-1 Quantikine ELISA Kit (DET100):

Refer to the product for complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.
  1.   Prepare all reagents, standard dilutions, and samples as directed in the product insert.
  2.   Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
    3. 150 µL Assay Diluent
  3.   Add 150 µL of Assay Diluent to each well.
    4. 75 µL Standard, Control, or Sample
  4.   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.
  5.   Aspirate each well and wash, repeating the process 3 times for a total of 4 washes.
    6. 200 µL Conjugate
  6.   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.
  7.   Aspirate and wash 4 times.
    8. 200 µL Substrate Solution
  8.   Add 200 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes on the benchtop. PROTECT FROM LIGHT.
    9. 50 µL Stop Solution
  9.   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.

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