ADIPOQ ELISA Kit

Produktnummer ABIN612733

Produktdetails ADIPOQ ELISA Kit

Target: ADIPOQ (Adiponectin (ADIPOQ))

Reaktivität: Maus

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Untere Nachweisgrenze: 0.7 ng/mL

Applikation: ELISA

Verwendungszweck: The AssayMax Mouse Adiponectin ELISA Kit is designed for detection of adiponectin in mouse urine, plasma, serum and cell culture supernatants

Marke: AssayMax

Proben: Plasma, Cell Culture Supernatant

Analytische Methode: Quantitative

Spezifität: This assay recognizes both natural and recombinant mouse adiponectin. It can detect both globular domain and full-length adiponectin.

Bestandteile: Adiponectin Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against mouse adiponectin. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes, which can be cut to fit the format of the individual assay. Adiponectin Standard: Mouse adiponectin in a buffered protein base (100 ng, lyophilized). 1 Biotinylated Adiponectin Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against adiponectin (140µl). 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, 2 bottles). 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).

Benötigtes Material: 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.

Anwendungsinformationen

Probenmenge: 50 μL

Testdauer: 4 h

Plattentyp: Pre-coated

Protokoll: This assay employs a quantitative sandwich enzyme immunoassay technique that measures adiponectin in 4 hours. A polyclonal antibody specific for adiponectin has been pre-coated onto a microplate. Adiponectin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for adiponectin, 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.

Aufbereitung der Reagenzien:

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. 2 MIx Diluent Concentrate (10x): Dilute the MIx Diluent Concentrate 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Adiponectin Standard: Reconstitute the 100 ng of mouse adiponectin Standard with 2 ml of MIx Diluent to generate a standard solution of 50 ng/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 ng/ml) twofold with equal volume of MIx Diluent to produce 25, 12.5, 6.25, 3.125, 1.56 and 0.78 ng/ml. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [Adiponectin] (ng/ml) P1 1 part Standard (50 ng/ml) 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 Adiponectin Antibody (50x): Spin down the biotinylated antibody briefly and dilute the desired amount of the antibody 1:50 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.

Probennahme: 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 1:400 into MIx Diluent. The undiluted samples can be stored 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 1:400 into MIx Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. 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. Dilute samples 1:10 into MIx Diluent. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles. Urine: Collect urine using sample pot. Centrifuge samples at 600 x g for 10 minutes and assay. Dilute samples 1:2 into MIx Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.

Testdurchführung:

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 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 Biotinylated Adiponectin Antibody to each well and incubate for one hour. Wash the microplate as described 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 the microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 10 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.

Ergebnisberechnung:

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 dilution factor. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.

Testpräzision: Intra-assay and inter-assay coefficients of variation were 4.4 % and 7.2% respectively.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Prepare all reagents (working diluent buffer, wash buffer, standards, biotinylated- antibody, 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 and the biotinylated-antibody vial before opening and using contents. The kit should not be used beyond the expiration date.

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date. Store SP Conjugate and Biotinylated Antibody 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 at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

Referenzen für ADIPOQ ELISA Kit (ABIN612733)

Kim, Mitchell, Bursill, Sowers, Thetford, Cook, van Reyk, Davies: "The nitroxide radical TEMPOL prevents obesity, hyperlipidaemia, elevation of inflammatory cytokines, and modulates atherosclerotic plaque composition in apoE-/- mice." in: Atherosclerosis, Vol. 240, Issue 1, pp. 234-41, (2015) (PubMed).

Moreno-Navarrete, Moreno, Vidal, Ortega, Serrano, Xifra, Ricart, Fernández-Real: "Deleted in breast cancer 1 plays a functional role in adipocyte differentiation." in: American journal of physiology. Endocrinology and metabolism, Vol. 308, Issue 7, pp. E554-61, (2015) (PubMed).

Huang, Yang, Chan, Wang, Huang, Wu, Tsai, Yang, Liu: "Arsenic Exposure and Glucose Intolerance/Insulin Resistance in Estrogen-Deficient Female Mice." in: Environmental health perspectives, (2015) (PubMed).

Wang, Chang, Lee, Shaw: "trans-10,cis-12 Conjugated linoleic acid specifically increases tissue α-tocopherol mediated by PPARγ inhibition in mice." in: International journal of food sciences and nutrition, pp. 1-7, (2014) (PubMed).

Motawi, Bustanji, El-Maraghy, Taha, Al Ghussein: "Naproxen and Cromolyn as New Glycogen Synthase Kinase 3β Inhibitors for Amelioration of Diabetes and Obesity: An Investigation by Docking Simulation and Subsequent In Vitro/In Vivo Biochemical Evaluation." in: Journal of biochemical and molecular toxicology, (2013) (PubMed).

Bermejo-Alvarez, Rosenfeld, Roberts: "Effect of maternal obesity on estrous cyclicity, embryo development and blastocyst gene expression in a mouse model." in: Human reproduction (Oxford, England), (2012) (PubMed).

Tsao, Lodish, Fruebis: "ACRP30, a new hormone controlling fat and glucose metabolism." in: European journal of pharmacology, Vol. 440, Issue 2-3, pp. 213-21, (2002) (PubMed).

Details zu ADIPOQ

Target: ADIPOQ (Adiponectin (ADIPOQ))

Andere Bezeichnung: Adiponectin (ADIPOQ Produkte)

Hintergrund: Adiponectin, also known as Adipocyte Complement-Related Protein of 30kDa (ACRP30), is a secreted serum protein expressed exclusively in differentiated adipocytes. Studies indicated that decreased plasma adiponectin concentration is associated with obesity, insulin resistance , essential hypertension , inflammation and atherosclerosis , and acute myocardial infarction. On the other hand, increased adiponectin level leads to nephrotic syndrome.