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|Antigen||Interleukin 1, beta (IL1B) ELISA Kits|
|Reactivity||Rhesus Monkey Alternatives|
Kits with alternative reactivity to:
|Method Type||Sandwich ELISA|
|Detection Range||7.81 pg/mL - 500 pg/mL|
|Minimum Detection Limit||7.81 pg/mL|
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Product Details IL1B ELISA KitTarget Details Application Details Handling Images
|Purpose||The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of IL1b in simian serum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids.|
|Sample Type||Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate|
This assay has high sensitivity and excellent specificity for detection of Interleukin 1 Beta (IL1b).
|Cross-Reactivity (Details)||No significant cross-reactivity or interference between Interleukin 1 Beta (IL1b) and analogues was observed.|
|Material not included||
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|Alternative Name||Interleukin 1 Beta (IL1b) (IL1B ELISA Kit Abstract)|
|Pathways||NF-kappaB Signaling, Interferon-gamma Pathway, Negative Regulation of Hormone Secretion, Cellular Response to Molecule of Bacterial Origin, Carbohydrate Homeostasis, Glycosaminoglycan Metabolic Process, Myometrial Relaxation and Contraction, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Autophagy|
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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 Interleukin 1 Beta (IL1b). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Interleukin 1 Beta (IL1b). 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 Interleukin 1 Beta (IL1b), 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 Interleukin 1 Beta (IL1b) in the samples is then determined by comparing the O.D. of the samples to the standard curve.|
Serum: Use a serum separator tube and 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 freshly prepared serum immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2 - 8 °C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.
Tissue homogenates: The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed in ice-cold PBS (0.01 mol/L,pH 7.0-7.2) to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces and homogenized them in 5-10mL of PBS with a glass homogenizer on ice(Micro Tissue Grinders woks, too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugated for 5 minutes at 5000×g. Remove the supernate and assay immediately or aliquot and store at ≤-20 °C.
Cell Lysates:Cells must be lysed before assaying according to the following directions.
1. Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
2. Wash cells three times in cold PBS.
3. Resuspend cells in PBS (1×) and the cells was subject to ultrasonication for 4 times (or Freeze cells at ≤ -20 °C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.)
4. Centrifuge at 1500×g for 10 minutes at 2 - 8 °C to remove cellular debris.
Cell culture supernates and other 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 IL1b 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.
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Interleukin 1 Beta (IL1b) were tested 20 times on one plate, respectively.
|Restrictions||For Research Use only|
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|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.
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.
|Expiry Date||6 months|
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