Resistin Kit ELISA

N° du produit ABIN612746

Détail du produit Resistin Kit ELISA

Antigène: Resistin (RETN)

Reactivité: Souris

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Seuil minimal de détection: 20 pg/mL

Application: ELISA

Fonction: The AssayMax Mouse Resistin ELISA kit is designed for detection of Mouse resistin in plasma, tissue, and cell culture supernatants

Marque: AssayMax

Type d'échantillon: Plasma, Cell Culture Supernatant

Analytical Method: Quantitative

Réactivité croisée (Details): No significant cross-reactivity or interference was observed.

Ingrédients: Resistin Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody against resistin. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Resistin Standard: Mouse resistin in a buffered protein base (8 ng, lyophilized). Biotinylated resistin Antibody (100x): A 100-fold concentrated biotinylated polyclonal antibody against Mouse resistin (80µl). MIx Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml). 1 Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).

Matériel non inclus: Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water

Information d'application

Volume d'échantillon: 50 μL

Durée du test: 5 h

Plaque: Pre-coated

Protocole: This assay employs a quantitative sandwich enzyme immunoassay technique which measures resistin in 5 hours. A murine monoclonal antibody specific for resistin has been pre-coated onto a microplate. Resistinin standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for resistin, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Préparation des réactifs:

Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. MIx Diluent Concentrate (10x): Dilute MIx Diluent Conc. 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 8 ng of Mouse Resistin Standard with 2 ml of MIx Diluent to generate a standard solution of 4 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare triplicate standard points by serially diluting the Resistin standard solution (4 ng/ml) twofold with equal volume of MIx Diluent to produce 2, 1, 0.5, 0.25, 0.125, and 0.0625 ng/ml solutions. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [M. Resistin] (ng/ml) P1 1 part Standard (4 ng/ml) 4.000 P2 1 part P1 + 1 part MIx Diluent 2.000 P3 1 part P2 + 1 part MIx Diluent 1.000 P4 1 part P3 + 1 part MIx Diluent 0.500 P5 1 part P4 + 1 part MIx Diluent 0.250 P6 1 part P5 + 1 part MIx Diluent 0.125 P7 1 part P6 + 1 part MIx Diluent 0.063 P8 MIx Diluent 0.000 Biotinylated Resistin Antibody (100x): Spin down the briefly and dilute the desired amount of the antibody 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute Wash Buffer 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C.

Prélèvement de l'échantillon: Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Dilute samples 1:50 with MIx Diluent. Store the remaining samples at -20°C or below. Avoid repeated freeze-thaw cycles. (EDTA or Heparin can also be used as anticoagulant.) Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and assay. Dilute samples 1:50 into MIx Diluent. Store serum at -20°C or below. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles.

Procédure de l'essai:

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Standard or sample per well. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to complete remove liquid at each step. Add 50 µL of Biotinylated Resistin Antibody to each well and incubate for two hours. Wash five times with 200 µL of Wash Buffer as above. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash five times with 200 µL of Wash Buffer as above. Add 50 µL of Chromogen Substrate per well and incubate for about 13 minutes or till the optimal blue color density develops. Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.

Calcul des résultats:

Calculate the mean value of the triplicate readings for each standard and sample. To generate a Standard Curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. 3 Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

Précision du teste: Intra-assay and inter-assay coefficients of variation were 4.1 % and 7.5% respectively.

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: The kit should not be used beyond the expiration date.

Stock: 4 °C/-20 °C

Stockage commentaire: Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened MIx Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.

Références pour Resistin Kit ELISA (ABIN612746)

Plante, Menaouar, Danalache, Yip, Broderick, Chiasson, Jankowski, Gutkowska: "Oxytocin treatment prevents the cardiomyopathy observed in obese diabetic male db/db mice." dans: Endocrinology, Vol. 156, Issue 4, pp. 1416-28, (2015) (PubMed).

Détail du antigène

Antigène: Resistin (RETN)

Autre désignation: Resistin (RETN Produits)

Synonymes: RETN Kit ELISA, ADSF Kit ELISA, FIZZ3 Kit ELISA, RETN1 Kit ELISA, RSTN Kit ELISA, XCP1 Kit ELISA, Fizz3 Kit ELISA, Rstn Kit ELISA, Xcp4 Kit ELISA, resistin Kit ELISA, RETN Kit ELISA, Retn Kit ELISA

Sujet: Resistin, a novel adipose-derived protein, has been proposed to cause insulin-resistant states in obesity. Resistin is produced by white and brown adipose tissues but has also has been identified in several other tissues, including the hypothalamus, pituitary and adrenal glands, pancreas, gastrointestinal tract, myocytes, spleen, white blood cells and plasma. Resistin antagonizes insulin action, and is down regulated by rosiglitazone and peroxisome proliferator- activated receptor gamma agonists. Resistin is elevated in patients with type 2 diabetes and may play a role in the vascular complications of this disorder. Recently, Resistin has been discussed controversially as a missing link between obesity and insulin resistance

Pathways: Feeding Behaviour, Smooth Muscle Cell Migration