Interleukin 1beta, Human, Low Range (IL-1b) BioAssay ELISA Kit

Interleukin 1 (IL-1) is considered the first of the super-family of regulatory and inflammatory cytokines. 1 There are two distinct IL-1 proteins, Interleukin 1b and Interleukin 1a. Il-1 has a number of alternative names, including lymphocyte activating factor, endogenous pyrogen, catabolin, hemopoietin-1, melanoma growth inhibition factor, and osteoclast activating factor (2, 3). The properties and biological activities of IL-1 have been extensively reviewed. IL-1 is expressed by many cells and has multiple functions including local inflammation. Cells known to express IL-1b include astrocytes (6), adrenal cortical cells, 7 NK cells (8), macrophages and monocytes (9), endothelial cells (10), keratinocytes (16), mega-karyocytes and platelets (11, 12), neurons (13), neutrophils (14). oligodendroglia (6), osteoblasts (5), Schwann cells (6), trophoblasts (8) ,and T cells plus fibroblasts (4). , IL-1 has multiple functions including local inflammation. Following bacterial or immunoglobulin ligation of monocyte/macrophage CD14 (the LPS receptor) (17) or CD64 (the IgG receptor), (18) IL-1 can be released into a local environment. Within this environment, IL-1 impacts a number of cells. First, capillary endothelial cells are induced to do two things, one, secrete chemokines such as MCP-1 (19), and two, up-regulate the expression of vascular adhesion molecules such as E-Selectin, ICAM-1 and VCAM-1.20 MCP-1 provides a stimulus for chemotaxis and activates mononuclear cell integrins (21), thus facilitating mononuclear infiltration into an area of early inflammation. IL-1 also induces expression of itself in newly arriving monocytes, thus reinforcing the overall process (22). In terms of other pro-inflammatory molecules, IL-1 apparently is needed for the efficient production of IFN-g. On resident NK cells, IL-1 apparently works in conjunction with macrophage-derived IL-12 to induce IFN-g secretion ( 23), resulting in an IFN-g induced activation of macrophages (24). Finally, IL-1 also induces the expression of MMPs from resident fibroblasts. This can have at least two effects, first extracellular matrix degradation can facilitate monocyte migration, and second, MMPs are known to degrade IL-1b, thus down-modulating the local inflammatory response initiated by IL-1. IL-1 is generally thought of as prototypical pro-inflammatory cytokines (2, 3). The effects of IL-1, however, are not limited to inflammation, as IL-1 has also has been associated with bone formation and re-modeling (5, 25), insulin secretion (26, 27), appetite regulation (28, 29), fever induction, neuronal phenotype development, and IGF/GH physiology (30-32). , This IL-1b ELISA is a 3.5 hour solid phase immunoassay readily applicable to measure , IL-1b levels in serum, plasma, cell culture supernatant, and other biological fluids in the range of 0 to 400pg/ml. It showed no cross reactivity with various other cytokines superfamily proteins. This IL-1b ELISA is expected to be effectively used for further investigations into the relationship between IL-1b and various diseases. Principle of the Assay: This IL-1b 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-1b. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated monoclonal antibody preparation specific for IL-1b and incubated. IL-1b 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-1b and other components of the sample. In order to quantitate the amount of IL-1b 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 have 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-1b, 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-1b 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-1b concentration (pg/ml). The concentration of IL-1b in the samples is then determined by comparing the O.D. of the samples to the standard curve., , Sensitivity: <2pg/ml, Range: 2-400pg/ml , Kit Components:, 1. I7663-14A: Microtiter Plate 1x96wells, Pre-coated with anti-human IL-8 monoclonal antibody, 2. I7663-14B: IL-1b Mab (Biotin) 1x7ml, Anti-human IL-1b monoclonal antibody conjugated to Biotin, Supplied as a liquid., 3. I7663-14C: Avidin (HRP) 1x12ml, Avidin conjugated to horseradish peroxidase, Supplied as a liquid, 4. I7663-14D: IL-1b Standard, Recombinant 2x800pg, Recombinant human IL-1b in a buffered protein base with preservative., Supplied as a lyophilized powder, 5. I7663-14E: Calibrator Diluent I 1x25ml, Supplied as animal serum with buffer and preservative. For serum/plasma testing, 6. I7663-14F: Calibrator Diluent II 1x25ml, Supplied as celll culture medium with calf serum and preservative. For cell culture supernatant testing, 7. I7663-14G: Wash Buffer (20X) 1x60ml, Supplied as a concentrated solution of buffered surfactant, 8. I7663-14H: Substrate A 1x10ml, Supplied as a buffered solution containing hydrogen peroxide, 9. I7663-14J: Substrate B 1x10ml, Supplied as a buffered solution containing TMB, 10. I7663-14K: Stop Solution 1x14ml, Supplied as a liquid containing 2N sufuric acid , Storage and Stability: Store all kit components except I7663-14D at 4°C. Stable for 6 months. Store I7663-14D powder at 4°C and liquid at -20°C (30 days). For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.