Background: Inflammation is body's protective response to foreign subjects, pathogens, tissue damage, autoimmune and other harmful stimuli. It is initiated by production of a cascade of chemicals and cytokines in affected area. These pro-inflammatory mediators result in vasodilation, increased vascular permeability, influx of blood, plasma leakage, neutrophil and macrophage infiltration and activation. Inflammatory reaction plays an important role in limiting foreign substance and engulfing pathogens and tissue debris. Acute inflammation is the initial phase to eliminate invaded foreign substances, pathogens and other harmful stimuli. If the stimuli persist, chronic inflammation will evolve. Progressive tissue destruction and shifting cell types occurs simultaneously during chronic inflammation. The cytokine profiles that regulate the procedure are different depending on the causes, location and progress of the inflammation. IL-1alpha is constitutively produced as precursor by epidermal cells at large amount in healthy individual. It is likely that the cytokine is secreted through microvesicle formation since the precursor doesn't have a signal peptide. Both the unprocessed form and processed form of IL- 1alpha possess biological activity. IL-1alpha is important for controlling the invasion of pathogen through skin and wound healing. IL-1alpha has been found to stimulate its own production, fibroblast proliferation and collagen production, increase neutrophil count in blood, stimulate IL-2 production, B lymphocyte proliferation and maturation, increase the concentration of copper and lower the iron and zinc level, stimulate hepatocytes to produce acute phase protein, and act in synergy with TNF-alpha to stimulate the production of GM-CSF, G-CSF and IL-6. IL-1alpha was also found to contribute to the generation of type IV hypersensitivity reactions. IL-1beta has been found to shares the IL-1R receptor with other cytokines in IL-1 family. It is produced by activated macrophage as a proprotein, which is cleaved by caspase I to become active. Like IL-1alpha, IL-1beta is an important pro-inflammatory mediator. IL-1beta can stimulate the production of IL-6 and TNF-alpha. Persistent IL-1beta signaling was found to contribute to the chronic inflammatory reaction in brain by sustained activation of NFkappaB in human glial cells, which leads to prolonged induction of selective pro-inflammatory genes. IL-6, also called B-cell stimulatory factor-2 (BSF-2) and interferon beta-2, plays an essential role in the final differentiation of B-cells into immunoglobulin-secreting cells. IL-6 can be produced by macrophage through Toll-like receptors in response to pathogenic molecular stimuli. Toll-like receptors are pattern recognition receptors recognize pathogen associated molecule patterns. IL-6 causes increased body temperature in acute inflammatory phase by initiating synthesis of Prostaglandin E2 in hypothalamus. The cytokine is also involved in inducing myeloma and plasmacytoma growth, nerve cell differentiation, and acute phase reactant production. Like IL-6, IL-8 can be secreted by cells with Toll-like receptors in response to the stimulation of pathogens. IL-8's primary function is to recruit neutrophils and other target cells through chemotaxis. Neutrophils, then, phagocytose the antigen which triggers the antigen pattern Tolllike receptors. GM-CSF is an extracellular homodimer polyprotein, functioning as a hematopoietic growth factor and immune modulator. It can be produced and act upon a variety of cell types, including T-lymphocytes, B-lymphocytes, monocytes/macrophages, endothelial cells, fibroblasts, stromal cells, mesothelial cells, kerotinocytes, osteoblasts, uterine epithelial cells, synoviocytes, mast cells and various solid tumors. GM-CSF stimulates stem cells to produce granulocytes and monocytes to cope with infection. Recombinant GM-CSF is used to boost white blood cell count of cancer patients after chemotherapy. rGM-CSF may also be useful as an immune tonic for anemia and AIDS patients. IFN-gamma is produced predominantly by natural killer and natural killer T cells as part of the innate immune response, and by CD4 and CD8 cytotoxic T lymphocyte (CTL), effector T cells once antigen-specific immunity develops. IFN-gamma primarily stimulates its own expression and upregulates other genes to stimulate and modulate immunity through the Jak-Stat signaling pathway. It promoters T helper 1 differentiation and cell associated immunity, and also suppresses T helper 2 differentiation and humoral immunity. IFN-gamma is released from viral infected cells and acts upon neighboring cells to produce large amount protein kinase R, which phosphorylates transcription initiation factor elF in response to viral infection. As a results, enzymes critical to mRNA replication is reduced and viral mRNA replication inhibited. In conjunction with CD40, IFN-gamma binds to and activates macrophages, which are then able to kill intracellular pathogens. Bound IFN-gamma causes the macrophage to produce elevated amounts of both MHC class I and II molecules, thus increasing the macrophage's presentation of foreign peptides. It also stimulates the production of antigenprocessing associated transportors and enzymes. IFN-gamma is critical for controlling viral and other intracellular pathogen infection and tumor development. Aberrant IFN-gamma expression is also associated with a number of autoimmune diseases MCAF is also called monocyte chemotactic protein-1 (MCP-1) and chemokine (C-C motif) ligand 2 (CCL2). It is primarily secreted by monocytes, macrophages and dendritic cells. It can be induced by platelet derived growth factor (PDGF). This cytokine displays chemotactic activity for monocytes and basophils but not for neutrophils or eosinophils. MCAF causes the degranulation of basophils and mast cells and augments the activity of monocyte and macrophage. MCAF plays an important role in inflammation, and also implicated with angiogenesis, auto-immune diseases, renal diseases, chronic infection and granuloma formation. Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic inflammatory cytokine. It has both growth promotion and inhibition effect to some cells. Secretion of the cytokine at low level is beneficial to body's normal function as it maintains homeostasis by regulating the body's circadian rhythm and promotes the remodeling or replacement of injured and senescent tissue by stimulating fibroblast growth. TNF-alpha plays a role in the immune response to bacterial, and certain fungal, viral, and parasitic invasions as well as in the necrosis of specific tumors. In the local inflammatory immune response, TNF-alpha initiates a cascade of cytokines and increases vascular permeability, thereby recruiting macrophage and neutrophils to a site of infection. TNF-alpha also stimulates blood clotting which serves to contain the infection. TNF-alpha hyper-expression in response to the components of some bacteria such as LPS can cause septic shock.

Principle of the Assay: This enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microwells on the 8-well strips enclosed in the kit have been precoated with monoclonal antibodies specific to IL-1alpha, IL-1beta, L-6, IL-8, IFN-gamma, GM-CSF, MCAF, and TNF-alpha respectively. Standards or samples are then added to the strips, and the biotinconjugated detection antibody mixture will be added late on. The above cytokines, 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 components of the sample. In order to quantitatively determine the amount of cytokine 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. 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 coating antibody and the specific cytokine, biotin-conjugated antibody and enzyme-conjugated Avidin will develop a blue colour. The intensity of colour development is proportional to the concentration of the specific cytokine presented in the each wells. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour will change to yellow. The intensity is measured spectrophotometrically at a wavelength of 450nm ± 2 nm. Samples were tested together with standards diluted with a similar matrix, or one of the Calibrator Diluent provided with the kit. This allows the operator to produce Optical Density (O.D) versus cytokine concentration (pg/mL). The concentration of cytokines in the samples is then determined by comparing the O.D. of the samples to the standards.

Intended Uses: This multiplex ELISA kit for pro-inflammatory cytokines is designed for semi-quantitative and simultaneous determination of pro-inflammatory cytokines including interleukin-1alpha (IL-1alpha), interleukin 1beta (IL-1?), interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte macrophage colony stimulating factor (GM-CSF), monocyte chemotactic and activating factor (MCAF), and tumor necrosis factor-alpha (TNF-alpha) in cell culture supernatant and other biological samples. The kit is intended FOR LABORATORY RESEARCH USE ONLY and is not for use in test or therapeutic procedures.

Small volumes of Inflammatory Cytokine elisa kit vial(s) may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container`s cap. Certain products may require to ship with dry ice and additional dry ice fee may apply.

MBS590064 is a ready-to-use microwell, strip-or-full plate ELISA (enzyme-linked immunosorbent assay) Kit for analyzing the presence of the Inflammatory Cytokine, ELISA Kit target analytes in biological samples. The concentration gradients of the kit standards or positive controls render a theoretical kit detection range in biological research samples containing Inflammatory Cytokine. The ELISA analytical biochemical technique of the MBS590064 kit is based on Inflammatory Cytokine antibody-Inflammatory Cytokine antigen interactions (immunosorbency) and an HRP colorimetric detection system to detect Inflammatory Cytokine antigen targets in samples. The ELISA Kit is designed to detect native, not recombinant, Inflammatory Cytokine. Appropriate sample types may include undiluted body fluids and/or tissue homogenates, secretions. Quality control assays assessing reproducibility identified the intra-assay CV (%) and inter-assay CV(%).