Endothelin 1 Kit ELISA

N° du produit ABIN2815086

Détail du produit Endothelin 1 Kit ELISA

Antigène: Endothelin 1 (EDN1)

Reactivité: Humain

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Application: ELISA

Fonction: The DetectX® Endothelin-1 (ET-1) kit is designed to quantitatively measure ET-1 present in a vari- ety samples and tissue culture media.

Marque: DetectX®

Type d'échantillon: Serum, Plasma, Tissue Culture Medium

Analytical Method: Quantitative

Réactivité croisée (Details): The following cross reactants were tested in the assay and calculated at the 50 % binding point. Steroid Cross Reactivity: Endothelin-1 (human, bovine, porcine, dog, rat, mouse), 100 % Endothelin-3 (human, bovine, porcine, dog, rat, mouse), 6.8 % Big Et-1 (human <0.04 %)

Homologie: Mouse (Murine), Cow (Bovine), Rat (Rattus), Dog (Canine), Pig (Porcine)

Ingrédients: Clear Coated 96 Well Plate One Plate Clear plastic microplate with break-apart strips coated with monoclonal antibody to human Endothe- lin-1.
Endothelin-1 Standard 50 μL Endothelin-1 at 10 ng/mL in a special stabilizing solution.
DetectX® Endothelin-1 Conjugate 5 mL An antibody to human Endothelin-1 labeled with peroxidase.
Assay Buffer Concentrate 28 mL A 5X concentrate that should be diluted with deionized or distilled water.
Extraction Solution 50 mL A solution used to extract Endothelin-1 from samples.
Wash Buffer Concentrate 30 mL A 20X concentrate that should be diluted with deionized or distilled water.
TMB Substrate 11 mL
Stop Solution 5 mL A 1N hydrochloric acid solution. Caustic.
Plate Sealer 2 each

Matériel non inclus: Distilled or deionized water.
A Speedvac or other centrifugal evaporator, or a manifold and inert gas supply such as nitrogen to evaporate extracted samples.
Polypropylene or glass test tubes.
Repeater pipet and disposable tips capable of dispensing 100 and 50 μL.
A microplate washer.
Colorimetric 96 well microplate reader capable of reading optical density at 450 nm.
Software for converting raw relative optical density readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.
Contact your plate reader manufacturer for de- tails.

Information d'application

Indications d'application: This assay has been validated for human serum, plasma, and tissue culture media (TCM) samples only.
Samples containing visible particulate should be centrifuged prior to using.
Due to the high- ly conserved nature of endothelin-1 it is expected that this kit will measure human, bovine, porcine, dog, rat and mouse ET-1.
The end user should test this kit for application in their samples.

Plaque: Pre-coated

Protocole: An ET-1 standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
Standards or diluted samples are pipetted into a clear mi- crotiter plate coated with a monoclonal antibody to capture ET-1 present in the sample.
After a 60 minute incubation, the plate is washed and a peroxidase conjugated ET-1 antibody is added.
The plate is again incubated for 60 minutes and washed.
Substrate is then added to the plate, which reacts with the bound ET-1 conjugated antibody.
After a third incubation, the reaction is stopped and the intensity of the generated color is detected in a microtiter plate reader capable of measur- ing 450 nm wavelength.
The concentration of the ET-1 in the sample is calculated, after making suitable correction for dilution, using software available with most plate readers.

Préparation des réactifs:

Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine ET-1 concentrations.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Assay Buffer Dilute Assay Buffer Concentrate 1:5 by adding one part of the concentrate to four parts of deion- ized water.
Once diluted this is stable at 4 °C for 3 months.
Wash Buffer Dilute Wash Buffer Concentrate 1:20 by adding one part of the concentrate to nineteen parts of deionized water.
Once diluted this is stable at room temperature for 3 months.
Standard Preparation Label test tubes as #1 through #8.
Pipet 990 μL of Assay Buffer into tubes #1.
Pipet 150 μL of Assay Buffer into tubes #2 to #8.
The ET-1 stock solution contains an organic solvent.
Prerinse the pipet tip several times to ensure accurate delivery.
Carefully add 10 μL of the 10 ng/mL ET-1 standard to tube #1 and vortex completely.
Take 150 μL of the ET-1 solution in tube #1 and add it to tube #2 and vortex completely.
Repeat the serial dilutions for tubes #3 through #8.
The con- centration of ET-1 in the tubes #1 through #8 will be 100, 50, 25, 12.5, 6.25, 3.125, 1.563 and 0.781 pg/mL.
Use all Standards within 2 hours of preparation.

Préparation de l'échantillon:

Serum and Plasma Samples Serum and plasma samples should be extracted with the provided Extraction Solution, or with a solid phase C18 column extraction protocol prior to running in the kit. Protocol Using Extraction Solution: Mix 1 part sample with 1.5 parts of Extraction Solution. Vortex and then nutate at room temperature for 90 minutes. Centrifuge for 20 minutes at 4 °C at 1660 x g. Speedvac supernatant to dryness at 37 °C. Reconstitute sample with 250 µL of Assay Buffer. Use all samples within 2 hour of preparation.

Procédure de l'essai:

1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification. Determine the number of wells to be used and return unused wells to foil pouch with desiccant. Seal the ziploc plate bag and store at 4ºC.
   2. Pipet 50 μL of samples or standards into wells in the plate. Pipet 50 μL of Assay Buffer into the zero standard wells.
   3. Cover the plate with the plate sealer and incubate at room temperature for 60 minutes.
   4. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels.
   5. Add 50 μL of the DetectX® Endothelin-1 Antibody Conjugate to each well, using a repeater pipet.
   6. Cover the plate with the plate sealer and incubate at room temperature for 60 minutes.
   7. Aspirate the plate and wash each well 4 times with 300 μL wash buffer. Tap the plate dry on clean absorbent towels.
   8. Add 100 μL of the TMB Substrate to each well, using a repeater pipet.
   9. Incubate the plate at room temperature for 30 minutes. 10. Add 50 μL of the Stop Solution to each well, using a repeater pipet. 11. Read the optical density generated from each well at 450 nm. 12. Use the plate reader's built-in 4PLC software capabilities to calculate Endothelin-1 concentration for each sample.

Calcul des résultats:

Average the duplicate OD readings for each standard and sample.
Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic curve (4PLC) fit.
The sample concentrations should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from www.myassays.com/arbor-assays-endothelin-1-(et-1)-eia-kit.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. tyPical data Sample Mean OD Endothelin-1 Conc. (pg/mL) Standard 1 1.281 100 Standard 2 0.656 50 Standard 3 0.378 25 Standard 4 0.238 12.5 Standard 5 0.177 6.25 Standard 6 0.150 3.125 Standard 7 0.138 1.563 Standard 8 0.123 0.781 Zero 0.114 0 Sample 1 0.632 44.74 Sample 2 0.348 22.79 Always run your own standard curve for calculation of results.
Do not use this data.

Précision du teste: Three samples were diluted with Assay Buffer and run in replicates of 20 in an assay.
Inter Assay Precision:
Three samples were diluted with Assay Buffer and run in duplicates in eighteen assays run over multiple days by three operators.

Restrictions: For Research Use only

Stockage

Précaution d'utilisation: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
The antibody coated plate needs to be stored desiccated.
The silica gel pack included in the foil ziploc bag will keep the plate dry.
The silica gel pack will turn from blue to pink if the ziploc has not been closed properly.
This kit utilizes a peroxidase-based readout system.
Buffers, including other manufacturers Wash Buffers, containing sodium azide will inhibit color production from the enzyme.
Make sure all buffers used for samples are azide free.
Ensure that any plate washing system is rinsed well with deionized water prior to using the supplied Wash Buffer as prepared on Page 8.
The Stop Solution is acid.
The solution should not come in contact with skin or eyes.
Take appro- priate precautions when handling this reagent.

Stock: 4 °C,RT

Stockage commentaire: All components of this kit should then be stored at 4 °C until the expiration date of the kit.

Détail du antigène

Antigène: Endothelin 1 (EDN1)

Autre désignation: Endothelin-1 (ET-1) (EDN1 Produits)

Synonymes: et1 Kit ELISA, et-1 Kit ELISA, Edn1 Kit ELISA, EDN Kit ELISA, ET-1 Kit ELISA, ET1 Kit ELISA, PPET1 Kit ELISA, HDLCQ7 Kit ELISA, preproET Kit ELISA, Et1 Kit ELISA, fb14d01 Kit ELISA, wu:fb14d01 Kit ELISA, PPET-1 Kit ELISA, EDN1 Kit ELISA, edn1 Kit ELISA, edn1-a Kit ELISA, edn1-b Kit ELISA, endothelin 1 Kit ELISA, endothelin 1 S homeolog Kit ELISA, endothelin 1 L homeolog Kit ELISA, edn1 Kit ELISA, EDN1 Kit ELISA, Edn1 Kit ELISA, edn1.S Kit ELISA, edn1.L Kit ELISA

Sujet: Endothelin-1 (ET-1), a peptide of 21 amino acid residues, is a pleiotropic molecule known for its ac- tion as a potent vasoconstrictor (1). 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 amino acid 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 Big ET-1. Big ET-1 is then cleaved by the membrane-bound metalloprotease Endothelin- converting enzyme (ECE-1), producing the potent mature form, ET-1 (7, 8). The vascular endothe- lium is an abundant source of ET-1 (3, 9). It may also be expressed by leukocytes, smooth muscle cells, mesangial cells, cardiac myocytes, and astrocytes (10, 11). ET-1 can be induced in endothelial cells by many factors including mechanical stimulation, various hormones, and pro-inflammatory cytokines. Production is inhibited by nitric oxide (NO), cyclic nucleotides, prostacyclin, and atrial natriuretic peptide (ANP) (12-14). ET-1 also stimulates cardiac contraction and the growth of cardiac myocytes, regulates the release of vasoactive substances, and stimulates smooth muscle cell mitogenesis. ET-1 may control in- flammatory responses by promoting the adhesion and migration of neutrophils and stimulating the production of pro-inflammatory cytokines. It has also been implicated in cancer progression regulating the proliferation and migration of tumor cells and acting as a pro-angiogenic factor (17). ET-1 has putative roles in other pathologies including septic shock, atherosclerosis, heart fail- ure, renal insufficiency, pulmonary hypertension, and cerebrovascular conditions associated with subarachnoid hemorrhage (11)

Pathways: Hormone Transport, Negative Regulation of Hormone Secretion, Regulation of Systemic Arterial Blood Pressure by Hormones, cAMP Metabolic Process, Regulation of Muscle Cell Differentiation, Regulation of G-Protein Coupled Receptor Protein Signaling, Regulation of Cell Size