Albumin Kit ELISA

N° du produit ABIN612764

Détail du produit Albumin Kit ELISA

Antigène: Albumin (ALB)

Reactivité: Rat

Méthode de détection: Colorimetric

Type de méthode: Competition ELISA

Seuil minimal de détection: 0.7 µg/mL

Application: ELISA

Fonction: The AssayMax Rat Albumin ELISA (Enzyme-Linked Immunosorbent Assay) kit employs a quantitative competitive enzyme immunoassay technique that measures rat plasma and serum albumin

Marque: AssayMax

Type d'échantillon: Plasma

Analytical Method: Quantitative

Ingrédients: Rat Albumin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against rat albumin. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. 1 Rat Albumin Standard: Rat albumin in a buffered protein base (150 µg, lyophilized). Biotinylated Albumin: 1 vial, lyophilized. 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). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80µl). 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: 25 μL

Durée du test: < 2 h

Plaque: Pre-coated

Protocole: A polyclonal antibody specific for rat albumin has been pre-coated onto a 96-well microplate with removable strips. Albumin in standards and samples is competed with a biotinylated Albumin sandwiched by the immobilized antibody and 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 the MIx Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 150 g of albumin standard with 3 ml of MIx Diluent to generate a solution of 50 g/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (50 g/ml) 1:2 with MIx Diluent to generate 25, 12.5, 6.25, 3.13, 1.56 and 0.78 g/ml solutions. MIx Diluent serves as the zero standard (0 g/ml). Any remaining solution should be frozen at -20°C 2 Standard Point Dilution [R. Albumin] ( g/ml) Standard (100 g/ml) P1 50.00 P2 1 part P1 + 1 part MIx Diluent 25.00 P3 1 part P2 + 1 part MIx Diluent 12.50 P4 1 part P3 + 1 part MIx Diluent 6.25 P5 1 part P4 + 1 part MIx Diluent 3.13 P6 1 part P5 + 1 part MIx Diluent 1.56 P7 1 part P6 + 1 part MIx Diluent 0.78 P8 MIx Diluent 0.00 Biotinylated Albumin (2x): Dilute Biotinylated Albumin with 5 ml MIx Diluent to produce a 2-fold stock solution, which should be further diluted 1:2 with MIx Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 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 1:8000 into MIx Diluent. Store samples at -20°C or below for up to 3 months. 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 1:8000 into MIx Diluent. Store serum 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 25 µL of standard or sample per well, and immediately add 25 µL of Biotinylated Albumin to each well (on top of the Standard or sample) and mix gently. Cover wells with a sealing tape and incubate for one hour. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µL of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 7 minutes or till the optimal blue color density develops. Gently tap 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. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. 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 duplicate or 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. 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 4.7 % and 7.3 % respectively.

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: Prepare all reagents (working diluent buffer, wash buffer, standards, biotinylated- protein, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this protocol. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial before opening and using contents. The kit should not be used beyond the expiration date.

Stock: 4 °C/-20 °C

Stockage commentaire: Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date. Store SP Conjugate at -20°C Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C Opened unused microplate wells may be returned to the foil pouch with the desiccant packs. Reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator. Diluent (1x) may be stored for up to 1 month at 2-8°C. Store Standard and Biotinylated Protein at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

Références pour Albumin Kit ELISA (ABIN612764)

Xu, Jiang, Wang, Wang, Liu, Peng, Fang, Deng, You, Zhou, Zhang, Lou: "C-peptide ameliorates renal injury in type 2 diabetic rats through protein kinase A-mediated inhibition of fibronectin synthesis." dans: Biochemical and biophysical research communications, Vol. 458, Issue 3, pp. 674-80, (2015) (PubMed).

Al-Rasheed, Al-Rasheed, Attia, Al-Amin, Al-Ajmi, Hasan, Mohamad, Sinjilawi: "Renoprotective Effects of Fenofibrate via Modulation of LKB1/AMPK mRNA Expression and Endothelial Dysfunction in a Rat Model of Diabetic Nephropathy." dans: Pharmacology, Vol. 95, Issue 5-6, pp. 229-39, (2015) (PubMed).

Buyl, De Kock, Bolleyn, Rogiers, Vanhaecke: "Measurement of Albumin Secretion as Functionality Test in Primary Hepatocyte Cultures." dans: Methods in molecular biology (Clifton, N.J.), Vol. 1250, pp. 303-8, (2015) (PubMed).

Chen, Stead, Advani, Yaqoob, Thai, Kabir, Yuen, Connelly, Gilbert, Advani: "Hyperglycemia and renal mass ablation synergistically augment albuminuria in the diabetic subtotally nephrectomized rat: implications for modeling diabetic nephropathy." dans: Nephron extra, Vol. 2, Issue 1, pp. 115-24, (2012) (PubMed).

Détail du antigène

Antigène: Albumin (ALB)

Autre désignation: Albumin (ALB Produits)

Synonymes: PRO0883 Kit ELISA, PRO0903 Kit ELISA, PRO1341 Kit ELISA, ALB Kit ELISA, CSA Kit ELISA, Alb-1 Kit ELISA, Alb1 Kit ELISA, Albza Kit ELISA, LOC100136344 Kit ELISA, alb-a Kit ELISA, alb-b Kit ELISA, albumin Kit ELISA, serum albumin 1 Kit ELISA, albumin S homeolog Kit ELISA, ALB Kit ELISA, Alb Kit ELISA, LOC100136344 Kit ELISA, alb.S Kit ELISA

Sujet: Albumin is serum hepatic protein, the most abundant protein in serum and contributes to the maintenance of oncotic pressure as well as to transport of hydrophobic molecules. Serum albumin level has been linked in clinical practice to several diseases. Low albumin levels can suggest liver , kidneys disease , inflammation , shock , and malnutrition. On the other hand, high albumin levels usually reflect dehydration.

Pathways: Lipid Metabolism