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|Antigen||Tumor Necrosis Factor (TNF) ELISA Kits|
Kits with alternative reactivity to:
|Method Type||Sandwich ELISA|
|Detection Range||3.12-200 pg/mL|
|Minimum Detection Limit||3.12 pg/mL|
|20 references available|
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Product Details Tumor Necrosis Factor ELISA KitTarget details Application Details Handling ProductDetails: References for Tumor Necrosis Factor Kit (ABIN415985) Images
|Purpose||The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of TNFa in Serum,Plasma,Tissue Homogenate,Cell Lysate,Cell Culture Supernatant,Biological Fluids|
|Sample Type||Serum, Plasma, Tissue Homogenate, Cell Lysate, Cell Culture Supernatant, Biological Fluids|
This assay has high sensitivity and excellent specificity for detection of Tumor Necrosis Factor Alpha (TNFa).
|Cross-Reactivity (Details)||No significant cross-reactivity or interference between Tumor Necrosis Factor Alpha (TNFa) and analogues was observed.|
|Material not included||
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|Alternative Name||TNFa (TNF ELISA Kit Abstract)|
|Pathways||NF-kappaB Signaling, Apoptosis, Caspase Cascade in Apoptosis, Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Positive Regulation of Endopeptidase Activity, Hepatitis C, Protein targeting to Nucleus|
Application DetailsProduct Details Tumor Necrosis Factor ELISA Kit Target details Handling ProductDetails: References for Tumor Necrosis Factor Kit (ABIN415985) Images back to top
Information on standard material:
|Sample Volume||100 μL|
|Assay Time||3 h|
|Protocol||The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Tumor Necrosis Factor Alpha (TNFa). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Tumor Necrosis Factor Alpha (TNFa). 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 Tumor Necrosis Factor Alpha (TNFa), 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 450nm ± 10nm. The concentration of Tumor Necrosis Factor Alpha (TNFa) 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.
Cell Culture Supernatant: 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.
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 TNFa 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 Tumor Necrosis Factor Alpha (TNFa) were tested 20 times on one plate, respectively.
|Restrictions||For Research Use only|
HandlingProduct Details Tumor Necrosis Factor ELISA Kit Target details Application Details ProductDetails: References for Tumor Necrosis Factor Kit (ABIN415985) 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.|
The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5 % within the expiration date under appropriate storage condition.
|Expiry Date||6 months|
ProductDetails: References for Tumor Necrosis Factor Kit (ABIN415985)Product Details Tumor Necrosis Factor ELISA Kit Target details Application Details Handling Images back to top
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Ortencio, Renzo, Sobrinho B, Kobashigawa, Crivelaro, Silva, Aldrovani, Ribeiro, Mineo, Laus: "Effects of morphine on the expression of cytokines and inflammatory mediators in a rabbit model of endotoxin-induced experimental uveitis." in: Arquivos brasileiros de oftalmologia, Vol. 78, Issue 6, pp. 371-5, 2015
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Sakuramoto, Shimojo, Jesmin, Unoki, Kamiyama, Oki, Miya, Kawano, Mizutani: "Repeated open endotracheal suctioning causes gradual desaturation but does not exacerbate lung injury compared to closed endotracheal suctioning in a rabbit model of ARDS." in: BMC anesthesiology, Vol. 13, Issue 1, pp. 47, 2013
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