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|Antigen||Wingless-Type MMTV Integration Site Family, Member 5A (WNT5A) ELISA Kits|
|Reactivity||Human, Mouse (Murine), Rat (Rattus) Alternatives|
Kits with alternative reactivity to:
|Methode Type||Sandwich ELISA|
|Detection Range||0.15-10 ng/mL|
|Minimum Detection Limit||0.15 ng/mL|
|9 references available|
|Supplier||Log in to see|
Product Details WNT5A ELISA KitTarget details Application Details Handling References for WNT5A Kit (ABIN481725) Images
|Purpose||The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of WNT5A in Serum,Plasma,Tissue Homogenate,Cell Lysate,Biological Fluids|
|Sample Type||Tissue Homogenate, Cell Lysate, Cell Culture Supernatant, Biological Fluids|
|Specificity||This assay has high sensitivity and excellent specificity for detection of Wingless Type MMTV Integration Site Family, Member 5A (WNT5A).|
|Cross-Reactivity (Details)||No significant cross-reactivity or interference between Wingless Type MMTV Integration Site Family, Member 5A (WNT5A) and analogues was observed.|
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding two standard deviations - the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.
|Material not included||
Target detailsProduct Details WNT5A ELISA Kit Application Details Handling References for WNT5A Kit (ABIN481725) Images back to top
|Alternative Name||WNT5A (WNT5A ELISA Kit Abstract)|
Application DetailsProduct Details WNT5A ELISA Kit Target details Handling References for WNT5A Kit (ABIN481725) Images back to top
Information on standard material:
|Sample Volume||100 μL|
|Assay Time||4.5 h|
|Plate||Pre-coated,Strips (12 x 8)|
The microtiter plate provided in this kit has been pre-coated with an antibody specific to WNT5A. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for WNT5A.
Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated.
After TMB substrate solution is added, only those wells that contain WNT5A, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color.
The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 10 nm. The concentration of WNT5A in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Serum: Allow samples to clot for two hours at room temperature or overnight at 4°C before centrifugation for 20 minutes at approximately 1000 × g. Assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
Tissue Homogenates: The preparation of tissue homogenates will vary depending upon tissue type. For this assay, rinse tissues in ice-cold PBS (0.02mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weigh before homogenization. Mince the tissues to small pieces and homogenize them in 5-10 mL of PBS with a glass homogenizer on ice (Micro Tissue Grinders work, too). Sonicate the resulting suspension with an ultrasonic cell disrupter or subject it to two freeze-thaw cycles to further break the cell membranes. Centrifugate the homogenates for 5 minutes at 5000 × g. Remove the supernate and assay immediately or aliquot and store at -20°C
Cell Lysate: Cells must be lysed before assaying according to the following directions. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly). Wash cells three times in cold PBS. Resuspend cells in PBS (1×) and subject them to ultrasonication for 4 times (or Freeze cells at -20 °C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.) Centrifuge at 1500 × g for 10 minutes at 2 - 8°C to remove cellular debris.
Biological Fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
|Calculation of Results||
Average the duplicate readings for each standard, control, and samples and subtract the average zero standard optical density. Construct a standard curve by plotting the mean O.D. and concentration for each standard and draw a best fit curve through the points on the graph or create a standard curve on log-log graph paper with WNT5A concentration on the y-axis and absorbance on the x-axis. Using some plot software, for instance, curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the known concentration of the standard (Y-axis), although concentration is the independent variable and O.D. value is the dependent variable. However, the O.D. values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), plotting log of the data to establish standard curve for each test is recommended. Typical standard curve below is provided for reference only.
Intra-assay Precision (precision within an assay): 3 samples with low, middle and high level human WNT5A were tested 20 times on one plate, respectively.
Inter-assay Precision (precision between assays): 3 samples with low, middle and high level human WNT5A were tested on 3 different plates, 8 replicates in each plate.
CV (%) = SD/mean X 100
|Restrictions||For Research Use only|
HandlingProduct Details WNT5A ELISA Kit Target details Application Details References for WNT5A Kit (ABIN481725) Images back to top
|Precaution of Use||The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.|
|Handling Advice||To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.|
|Storage||4 °C/-20 °C|
|Expiry Date||6 months|
References for WNT5A Kit (ABIN481725)Product Details WNT5A ELISA Kit Target details Application Details Handling Images back to top
|Product cited in:||
Schulte, Kragelund, Müller, Hagen, Elke, Titz, Schädler, Schumacher, Weiler, Bewig, Schreiber, Laudes: "The wingless-related integration site-5a/secreted frizzled-related protein-5 system is dysregulated in human sepsis." in: Clinical and experimental immunology, Vol. 180, Issue 1, pp. 90-7, 2015
Chatani, Kamada, Kizu, Ogura, Furuta, Egawa, Hamano, Ezaki, Kiso, Shimono, Ouchi, Yoshida, Takehara: "Secreted frizzled-related protein 5 (Sfrp5) decreases hepatic stellate cell activation and liver fibrosis." in: Liver international : official journal of the International Association for the Study of the Liver, Vol. 35, Issue 8, pp. 2017-26, 2015
Xu, Xu, Zhao, Yang, Liu, Wen, Zhang: "Wnt5a reverses the inhibitory effect of hyperoxia on transdifferentiation of alveolar epithelial type II cells to type I cells." in: Journal of physiology and biochemistry, Vol. 71, Issue 4, pp. 823-38, 2015
Bougault, Briolay, Boutet, Pilet, Delplace, Le Goff, Guicheux, Blanchard, Magne: "Wnt5a is expressed in spondyloarthritis and exerts opposite effects on enthesis and bone in murine organ and cell cultures." in: Translational research : the journal of laboratory and clinical medicine, 2015
Prats-Puig, Soriano-Rodríguez, Carreras-Badosa, Riera-Pérez, Ros-Miquel, Gomila-Borja, de Zegher, Ibáñez, Bassols, López-Bermejo: "Balanced duo of anti-inflammatory SFRP5 and proinflammatory WNT5A in children." in: Pediatric research, Vol. 75, Issue 6, pp. 793-7, 2014
Tan, Wang, Chu, Liu, Yi, Xiao: "SFRP5 correlates with obesity and metabolic syndrome and increases after weight loss in children." in: Clinical endocrinology, Vol. 81, Issue 3, pp. 363-9, 2014
Catalán, Gómez-Ambrosi, Rodríguez, Pérez-Hernández, Gurbindo, Ramírez, Méndez-Giménez, Rotellar, Valentí, Moncada, Martí, Sola, Silva, Salvador, Frühbeck: "Activation of noncanonical Wnt signaling through WNT5A in visceral adipose tissue of obese subjects is related to inflammation." in: The Journal of clinical endocrinology and metabolism, Vol. 99, Issue 8, pp. E1407-17, 2014
Carstensen, Herder, Kempf, Erlund, Martin, Koenig, Sundvall, Bidel, Kuha, Roden, Tuomilehto: "Sfrp5 correlates with insulin resistance and oxidative stress." in: European journal of clinical investigation, Vol. 43, Issue 4, pp. 350-7, 2013
Schulte, Müller, Neumann, Oberhäuser, Faust, Güdelhöfer, Brandt, Krone, Laudes: "Pro-inflammatory wnt5a and anti-inflammatory sFRP5 are differentially regulated by nutritional factors in obese human subjects." in: PLoS ONE, Vol. 7, Issue 2, pp. e32437, 2012
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