Interleukin 16, Human (LCF, prIL-16, FLJ16806, FLJ42735, FLJ44234, HST19289 ) BioAssay(TM) ELISA Kit

Introduction:, Interleukin-16 (IL-16) discovered in 19821, known formerly as lymphocyte chemoattractant factor (LCF) is a lymphocyte chemoattractant factor of T cell origin with selective activity for CD4+ T cells. LCF was designated interleukin-16 (IL-16) in 1995. IL-16 is a product of CD8+ cells, CD4+ cells2, eosinophils3, mast cells and epithelial cells derived from asthmatics4. Expression of IL-16 in asthmatic epithelium correlates with the number of infiltrating CD4+ T cells5. , LCF was originally identified and purified from the supernatants of ConA-stimulated peripheral blood mononuclear cells (PBMC). In addition to its chemotactic activity, it is a competence growth factor selective for CD4+ T cells dependent on an interaction with CD4 for induction of functional activity. Its cDNA codes for a novel 14-kD protein into a homotetrameric form is required for induction of biologic activity. IL-16 appears in culture supernatants as a relative molecular mass (Mr) ~56,000 biologically active, non-covalently linked tetramer, but migrates in monomeric form in SDS PAGE. Eluted monomeric peptides are inactive but reaggregate to Mr 56,000 regaining biological activity6. The protein expressed from the IL-16 cDNA demonstrates all the functions and chemical features of the native protein, including an identical pI, and autoaggregation into functional tetramers. IL-16 is preformed and stored in biologically active form in CD8+. T cells from which it is secreted following stimulation with histamine via H2 type receptors. The secretory process occurs within 4 hrs and does not require transcription, translation, or new protein synthesis. IL-16's ability to inhibit the MLR and other antigen induced activation suggests that it may be useful in inhibiting allograft rejection. Because IL-16 selectively induces IL-2 responsiveness in CD4+ T cells it may be useful (along with IL-2) for selective CD4+ T cell immune reconstitution in individuals with lymphopenia, for example following chemotherapy or HIV-1 infection. In the latter circumstance, its ability to inhibit HIV transcription should provide a protective role if used as a therapy in HIV-1 infected individuals7. , Inhibitors of IL-16 chemotactic function may be useful in diseases in which it appears to play a prominent role in the inflammatory process. The presence of IL-16 early after antigen challenge in asthma8, the marked upregulation of IL-16 synthesis by epithelium of asthmatics along with the correlation of IL-16 protein with the number of infiltrating CD4+ T cells in the airways of human asthmatics suggest that therapeutics aims at blocking IL-16 synthesis or function may be valuable in this disease. This IL-16 ELISA is a 4.5 hour solid phase immunoassay readily applicable to measure IL-16 in serum, plasma, cell culture supernatant, and other biological fluids in the range of 0 to 3200pg/ml. It showed no cross-reactivity with other cytokines tested such as EGF, EPO, GM-CSF, IL-1b, IL-7, IL-8, IFN-g, MCAF, MCP-3, M-CSF, SAA, TGF-b, and TNF-a. Principle of the Assay:, This IL-16 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 for IL-16. Standards or samples are then added to the appropriate microtiter plate wells and incubated. IL-16 if present, will bind and become immobilized by the antibody pre-coated on the wells. The microtiter plate wells are thoroughly washed to remove any unbound IL-16 and other components of sample. In order to quantitate the amount of IL-16 present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody specific for IL-16 is added to each well to "sandwich" the IL-16 immobilized during the first incubation. The microtiter plate then undergoes a second incubation. The wells are thoroughly washed to remove all unbound HRP-conjugated antibodies and a TMB (3,3'5,5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate solution are allowed to react over a short incubation period. Only those wells that contain IL-16 and enzyme-substrate reaction will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change measured spectrophotometrically at a wavelength of 450nm ± 2nm. , In order to measure the concentration of IL-16 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-16 (pg/ml). The concentration of IL-16 in the samples is then determined by comparing the O.D. of the samples to the standard curve. Kit Components: 1. IL-16 Microtiter Plate, 1x96 wells 2. IL-16 Conjugate, 1x25ml , 3. IL-16 Standard, 2x1 vials, 4. Calibrator Diluent I, 1x 30ml , 5. Calibrator Diluent II, 1x 50ml , 6. Wash Buffer (20X), 1x 60ml , 7. Substrate A, 1x16ml , 8. Substrate B, 1x16ml , 9. Stop Solution, 1x14ml, 2N Sulphuric Acid (H2SO4). Sensitivity: <20ng/ml , Range: 20-3200ng/ml, Storage and Stabilty: Store components at -20°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.