IL-2 sRa ELISA KIT
INTENDED USE
This Human IL-2 sRa ELISA Kit is to be used for the in vitro quantitative determination of human interleukin-2 soluble receptor alpha (IL-2 sRα) concentrations in serum, plasma, cell culture supernatant, and other biological fluids. This IL-2 sRa ELISA Kit is intended FOR LABORATORY RESEARCH USE ONLY and is not to be used in diagnostic or therapeutic procedures.
INTRODUCTION
The biological function of IL-2 is obtained by binding to the specific interleukin-2 receptor (IL-2R). The IL-2R consists of three non-covalently linked chains, all of which are type I transmembrane proteins and include the α chain (IL-2Rα, p55), β chain (IL-2Rβ, p75), and γ chain (IL-2Rγ, p65). The α chain is cleaved from the cell surface via nonspecific proteolysis. IL-2Rα and IL-2Rβγ dimers bind to different residues on the IL-2 protein. The IL-2Rα complex displays low affinity and the IL-2Rαβ complex displays intermediate affinity for IL-2 binding. Both IL-2Rα and IL-2Rαβ complexes are unable to transduce a signal. The IL-2Rβγ complex has intermediate affinity for IL-2 binding and can transduce a signal with a relatively high concentration of IL-2. The IL-2Rαβγ trimer is the high-affinity receptor for IL-2 and can transduce a signal successfully.
Many cells are capable of expressing IL-2Rα including the antigen-activated T cells and B cells, and approximately 10% of natural killer (NK) cells, leukemia and lymphoma cells. When produced by activated T cells, the α chain is 10-20 folds in excess of the β and γ chain. A soluble IL-2Rα can be detected in tissue culture media of IL-2R+ cells and in the serum of experimental animals and humans undergoing an immune response. The major biological activities of IL-2R include promoting the proliferative expansion of T cells and NK cells upon activation, promoting the persistence of antigen-selected memory T cells, and promoting homeostasis of the immune system after it has successfully responded to an antigen. However, the biological activity of soluble IL-2Rα is unclear. It has been reported that elevated IL-2 sRα level is accompanied by increased T and B cell activation and immune system activation as observed in rheumatoid arthritis, systemic lupus erythematosis (SLE), some leukemias and lymphomas. Because of its low affinity, IL-2 sRα would be expected to be an inhibitor of IL-2.
PRINCIPLE OF THE ASSAY
IL-2 sRa ELISA Kit
IL-2 sRa ELISA Kit is to be used for the in vitro quantitative determination of human interleukin-2 soluble receptor alpha (IL-2 sRα) concentrations in serum, plasma, cell culture supernatant, and other biological fluids. This IL-2 sRa ELISA Kit is intended FOR LABORATORY RESEARCH USE ONLY and is not to be used in diagnostic or therapeutic procedures. This IL-2 sRα ELISA Kit is a ready-to-use 4.5-hour solid phase immunoassay capable of measuring IL-2 sRα levels in serum, plasma, cell culture supernatant, and other biological fluids in the range of 0 to 2000 pg/mL. This IL-2 sRα ELISA Kit assay has shown no cross-reactivity with other cytokines tested, and is expected to be used effectively for further investigations into the relationship between IL-2 sRα and the various conditions mentioned.
This IL-2 sRa ELISA Kit applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific to IL-2 sRα. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for IL-2 sRα and incubated. IL-2 sRα if present, will bind and become immobilized by the antibody pre-coated on the wells and then become “sandwiched” by biotin conjugate. The microtiter plate wells are thoroughly washed to remove unbound IL-2 sRα and other components of the sample. In order to quantitatively determine the amount of IL-2sRα 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, each having 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-2 sRα, 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 450nm ± 2nm.
In order to measure the concentration of IL-2 sRα in the samples this 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-2 sRα concentration (pg/mL). The concentration of IL-2 sRα in the samples is then determined by comparing the O.D. of the samples to the standard curve.
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