Multiplex Human Cytokine ELISA Kit (Inflammatory)

RACTIVITY	Human
SENSITIVITY	Refer to inset
ASSAY RANGE	Refer to inset
REAGENTS PROVIDED	Refer to inset

INTENDED USE

This multiplex ELISA kit for pro-inflammatory cytokines is designed for semi-quantitative and simultaneous determination of pro-inflammatory cytokines including interleukin-1α (IL-1α), interleukin 1β (IL-1β), 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-α (TNF-α) in cell culture supernatant and other biological samples. The kit is intended FOR LABORATORY RESEARCH USE ONLY and is not for use in diagnostic or therapeutic procedures.

INTRODUCTION

Inflammation is body’s protective response to foreign subjects, pathogens, tissue damage, auto-immune 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-1α 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-1α possess biological activity.  IL-1a 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 activated macrophage as a proprotein, which is cleaved by caspase I to become active. Like IL-1α, IL-1β is an important pro-inflammatory mediator.  IL-1b can stimulate the production of IL-6 and TNF-a.  Persistent IL-1b signaling was found to contribute to the chronic inflammatory reaction in brain by sustained activation of NFkB 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 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 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 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-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 bothMHCclass 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 infection and tumor development.  Aberrant IFN-γ expression is also associated with a number of autoimmune diseases

MCAFis 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.  MCAFcauses the degranulation of basophils and mast cells and augments the activity of monocyte and macrophage. MCAFplays 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-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 inflammatory 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. 

This ELISA assay is a 3.5 hour solid phase immunoassay readily applicable to measure the levels of eight cytokines in cell culture supernatant, and other biological fluids.  It showed no cross reactivity with various other proteins.  This Multiplex ELISA is expected to be effectively used for investigations into the relationship inflammatory cytokine expression and various experimental, pathological and physiological conditions.