IL-15 ELISA KIT
INTENDED USE
This Human IL-15 ELISA Kit is to be used for the in vitro quantitative determination of human interleukin 15 (IL-15) concentrations in serum, plasma, and cell culture supernatant. This IL-15 ELISA Kit is intended FOR LABORATORY RESEARCH USE ONLY and is not for use in diagnostic or therapeutic procedures.
INTRODUCTION
Interleukin-15 (IL-15), a modest 14-15 kDa glycoprotein, is produced by monocytes and dendritic cells and at maturity reaches 114 amino acid residues in length. IL-15 is a member of the four α helix bound cytokine family that includes IL-2, IL-3, IL-5, IL-6, IL-7 and IL-9. Although the structure of IL-15 has not been determined, it is predicted to be similar to IL-2 and other members of the four-helix bundle cytokine family. IL-15 mRNA has been discovered in the placenta, skeletal muscle, kidneys, lungs, heart, fibroblasts, epithelial cells, dendritic cells, and monocytes. Human IL-15 shares approximately 97% and 73% amino acid sequence identity with simian and mouse IL-15, respectively. Both human and simian IL-15 molecules are active with mouse cells.
The biological activities of IL-15 are comparable to IL-2: IL-15 induces the proliferation of activated CD4-CD8-, CD4+CD8+, CD4+, and CD8+ cells and dendritic epidermal T cells. IL-15 co-stimulates proliferation of B cells activated with immobilized anti-human IgM or phorbol ester, but has no stimulatory effect on resting B cells. In combination with recombinant CD40L, IL-15 is a potent inducer of polyclonal IgM, IgG1, and IgA secretion, but does not cause production of IgG4 or IgE. The development, survival, and activation of natural killer (NK) cells is related to IL-15. IL-15 also affects the non-lymphoid tissues, such as muscle, brain, microglia, and mast cells. Abnormal levels of IL-15 have been reported in rheumatoid arthritis, inflammatory bowel disease, and in diseases associated with the retroviruses HIV and HTLV-I.
PRINCIPLE OF THE ASSAY
IL-15 ELISA Kit
IL-15 ELISA Kit is to be used for the in vitro quantitative determination of human interleukin 15 (IL-15) concentrations in serum, plasma, and cell culture supernatant. This IL-15 ELISA Kit is intended FOR LABORATORY RESEARCH USE ONLY and is not for use in diagnostic or therapeutic procedures. This IL-15 ELISA Kit is a ready-to-use 4.5-hour solid phase immunoassay readily capable of measuring IL-15 levels in serum, plasma, cell culture supernatant, and other biological fluids in the range of 0 to 2000 pg/mL. This IL-15 ELISA Kit has shown no cross-reactivity with other cytokines tested, and is expected to be used effectively for further investigations into the relationship between IL-15 and the various conditions mentioned. This IL-15 ELISA Kit applies a technique called a quantitative sandwich immunoassay.
The microtiter plate provided in this IL-15 ELISA Kit has been pre-coated with a monoclonal antibody specific to IL-15. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for IL-15 and incubated. IL-15 if present, will bind and become immobilized by the antibody pre-coated on the wells and then be “sandwiched” by biotin conjugate. The microtiter plate wells are thoroughly washed to remove unbound IL-15 and other components of the sample. In order to quantitatively determine the amount of IL-15 present in the sample, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Avidin is a tetramer containing four identical subunits that each has a high affinity-binding site for biotin. The wells are thoroughly washed to remove all unbound HRP-conjugated Avidin and a TMB (3,3'5,5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain IL-15, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.
In order to measure the concentration of IL-15 in the samples, this IL-15 ELISA Kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing.) According to the testing system, the provided standard is diluted (2-fold) with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D) versus IL-15 concentration (pg/mL). The concentration of IL-15 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
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