Inflammatory Cytokine Panel (multiplex) - human
For the simultaneous quantitative determination of pro-inflammatory cytokines including IL-1α, IL-1β, IL-6, IL-8, IFN-γ, GM-CSF, MCAF and TNF-α in cell culture supernatant, serum, plasma and other tissue samples.
The Human Inflammatory Cytokine Panel (Multiplex) ELISA is designed semi-quantitative and simultaneous determination of pro-inflammatory cytokines including interleukin-1α (IL-1α), interleukin 1β (IL-1β), interleu- kin-6 (IL-6), interleukin-8 (IL-8), interferon-γ (IFN-γ), granulocyte macrophage colony stimulating factor (GM- CSF), monocyte chemotactic and activating factor (MCAF) and tumor necrosis factor-α (TNF-α) in cell culture supernatant, serum, plasma and other tissue samples.
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. 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-1α is constitutively produced as a precursor by epidermal cells at large amounts in healthy individuals. 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-1α possess biological activity. IL-1α is important for controlling the invasion of pathogen through skin and wound healing. IL-1α 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-α to stimulate the production of GM-CSF, G-CSF and IL-6. IL-1α was also found to contribute to the generation of type IV hypersensitivity reactions.
IL-1β has been found to shares the IL-1R receptor with other cytokines in IL-1 family. It is produced by acti- vated macrophages as a proprotein, which is proteolytically processed to its active form by caspase 1 (CASP1/ ICE). IL-1α, IL-1β is an important pro-inflammatory mediator. IL-1β can stimulate the production of IL-6 and TNF-α. Persistent IL-1β signaling was found to contribute to the chronic inflammatory reaction in the brain by sustained activation of NFκB in human glial cells, leading to prolonged induction of selective pro-inflammatory genes.
IL-6 also referred to as 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 recog- nition receptors that recognize pathogen associated molecule patterns. IL-6 causes increased body tempera- ture 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 Toll-like 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 (RGM-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-γ is produced predominantly by natural killer (NK) and natural killer T (NKT) cells as part of the innate im- mune response, and by CD4, Th1 and CD8 cytotoxic T lymphocyte (CTL) effector T cells once antigen-specific immunity develops. IFN-γ primarily stimulates its own expression and up-regulates 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-γ 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-γ binds to and activates macrophages, which are then able to kill intracellular pathogens. Bound IFN-γ 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 antigen-processing associated transportors and enzymes. IFN-γ is critical for controlling viral and other intracellular pathogen in- fection and tumor development. Aberrant IFN-γ 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.
TNF-α 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-α 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 inflamma- tory immune response, TNF-α initiates a cascade of cytokines and increases vascular permeability, thereby recruiting macrophage and neutrophils to a site of infection. TNF-α also stimulates blood clotting which serves to contain the infection. TNF-α hyper-expression in response to the components of some bacteria such as LPS can cause septic shock.
Sample Type: serum, plasma, cell culture supernates, tissue samples
Sample Size: 100 uL
Standard Curve Range: dependent on the cytokine
Assay Length: < 3.5 hours
How It Works
The Human Inflammatory Cytokine Panel (Multiplex) ELISA is designed to measure the levels of eight cytokines in cell culture supernatant, serum, plasma and other tissue samples in 3.5 hours. This ELISA is expected to be effectively used for investigations into the relationship inflammatory cytokine expression and various experimental, pathological and physiological conditions.
This ELISA applies a technique called a quantitative sandwich immunoassay. Monoclonal antibodies specific to IL-1α, IL-1β, L-6, IL-8, INF-γ, GM-CSF, MCAF, and TNF-α respectively has been pre-coated onto a microplate. Standards or samples are then added to the strips, and the biotin-conjugated detection antibody mixture will be added later 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 microplate 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 color. The intensity of color 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 color will change to yellow. The intensity is measured spectrophotometrically at a wave- length 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 concentra- tion (pg/mL). The concentration of cytokines in the samples is then determined by comparing the O.D. of the samples to the standards.