Human IL-15 ELISA Assay
The Human IL-15 ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 4 pg/mL
Dynamic Range: 15.625 – 500 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: Interleukin 15
Interleukin 15 (IL-15) is a 14 kDa cytokine that is structurally and functionally related to IL- 2. Mature human IL-15 shares 70% amino acid sequence identity with mouse and rat IL-15. Alternate splicing generates isoforms of IL-15 with either a long or short signal peptide (LSP or SSP), and the SSP isoform is retained intracellularly. IL-15 associates with IL-15 Rα in the endoplasmic reticulum, and this complex is expressed on the cell surface. The dominant mechanism of IL-15 action is known as transpresentation in which IL-15 and IL-15 Rα are coordinately expressed on the surface of one cell and interact with complexes of IL-2 Rβ/γc on adjacent cells. This enables cells to respond to IL-15 even if they do not express IL-15 Rα. Consistent with its shared use of IL-2 receptor subunits, IL-15 induces IL-2-like effects in lymphocyte development and homeostasis. It is particularly important for the maintenance and activation of NK cells and CD8+ memory T cells. IL-15 also exerts pleiotropic effects on other hematopoietic cells and nonimmune cells. Ligation of membrane associated IL-15/IL-15Rα complexes induces reverse signaling that promotes cellular adhesion, tyrosine phosphorylation of intracellular proteins, and cytokine secretion by the IL-15/IL-15Rα expressing cells.
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The Eagle Biosciences Human Interleukin 15 (IL-15) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-15 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-15 present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for IL-15 is added to the wells and binds to the combination of capture antibody- IL-15 in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of IL-15 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven IL-15 standard dilutions and IL-15 sample concentration determined.
- Prepare all reagents and working standards as directed in the previous sections.
- Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
- Add 100 µl of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
- Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µl) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
- Add 100 µl of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
- Repeat the aspiration/wash.
- Add 100 µl of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C Avoid placing the plate in direct light.
- Repeat the aspiration/wash.
- Add 100 µl of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length;610-650nm is acceptable)
Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.
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