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 degrees C. Stable for 6 months. Store I7663-14D powder at 4 degrees C and liquid at -20 degrees C (30 days). For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
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