Interleukin 15, Human (IL-15) BioAssay ELISA Kit

Interleukin 15 (IL-15) is a novel cytokine that shares many biological properties with, but lacks amino acid sequence homology to, IL-2. IL-15 was originally identified in media conditioned by a monkey kidney eipthelial cell line (CVI/EBNA) based on its mitogenic activity on the murine T cell line, CTLL-2. IL-15 was also independently discovered as a cytokine produced by a human adult T cell leukemia cell line (HuT-102) that stimulated T cell proliferation and was designated IL-T. Human, simian and mouse IL-15 cDNA, as well as human and mouse IL-15 genomic clones, have been isolated and characterized. The IL-15 cDNA clones from all three species encode a 162 amino acid (aa) residue precursor protein containing a 48 aa residue leader that is cleaved to generate the 114 aa residue mature IL-15. Human IL-15 shares approximately 97% and 73% sequence identity with simian and mouse IL-15, respectively. Both human and simian IL-15 are active on mouse cells. 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 has biological activities similar to IL-2 and has been shown to stimulate the growth of natural killer cells, activated peripheral blood T lymphocytes tumor infiltrating lymphocytes (TILs) and B cells. In addition, IL-15 has also been shown to be a chemoattractant for human blood T lymphocytes and to be able to induce lymphokine-activated killer (LAK) activity in NK cells as well as to be able to induce the generation of cytolytic effector cells. It is likely that additional, as yet unidentified, functions for IL-15 will be discovered in the future. This IL-15 enzyme linked immunosorbent assay (ELISA) 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-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 quantitate 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 color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 2nm. In order to measure the concentration of IL-15 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-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. Sensitivity: <10pg/ml, Assay Range: 30-1000 pg/ml , Kit Components:, 1. IL-15 Microtiter Plate, 1x96 wells , 2. Biotin Conjugate, 1x7ml , 3. Avidin Conjugate, 1x14ml , 4. IL-15 Standard, 2x1 vials , 5. Calibrator Diluent I, 1x22ml For serum/plasma testing., 6. Calibrator Diluent II, 1x 22ml For cell culture supernatant testing., 7. Wash Buffer (20X), 1x 60ml , 8. Substrate A, 1x10ml , 9. Substrate B, 1x10ml , 10. Stop Solution, 1x14ml, Storage and Stability: Store components at 4°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.