IL-5 ELISA KIT
INTENDED USE
This Human IL-5 ELISA Kit is to be used for the in vitro quantitative determination of human interleukin 5 (IL-5) concentrations in serum, plasma, cell culture medium, and urine. This IL-5 ELISA Kit is intended for LABORATORY RESEARCH ONLY and is not to be used for diagnostic or therapeutic purposes.
INTRODUCTION
Human IL-5 is a 135 amino acid (aa) polypeptide with a predicted mass of 12.5 kDa. It is secreted by a restricted number of mesenchymal cell types. In its native state, mature IL-5 is synthesized as a 115 aa, highly glycosylated 22 kDa monomer that forms a 40-50 kDa disulfide-linked homodimer. Although the content of carbohydrate is high, carbohydrate is not needed for bioactivity. Monomeric IL-5 has no activity and requires a homodimer for function, which is in contrast to the receptor-related IL-3 and GM-CSF cytokines that exist only as monomers. Just as one IL-3 and GM-CSF monomer binds to one receptor, one IL-5 homodimer is able to engage only one IL-5 receptor. It has been suggested that IL-5 (as a dimmer) undergoes a general conformational change after binding to one receptor molecule and this change precludes binding to a second receptor. Human and mouse mature IL-5 are 71% identical at the aa level. While mouse IL-5 is highly active on human cells, human IL-5 is only marginally active on mouse cells.
Although many cells contribute to inflammation, eosinophils are noted for their contribution to late phase allergic-type disorders. Eosinophils make up less than 10% of the circulation leukocyte population, yet they are known to be extremely important in the inflammatory response to allergic and parasitic agents. When activated within tissues, eosinophils release highly basic preformed mediators such as eosinophil peroxidase and major basic protein. These substances are toxic to parasites and damaging to the surrounding tissue functionality including smooth muscle constriction, vascular permeability and superoxide-mediated tissue destruction. While eosinophils have been associated with these inflammatory reactions, the soluble mediators that influence eosinophil availability and function have only recently been identified. Interleukin 5 (IL-5) along with the chemokine eotaxin are key players in the coordination of the eosinophil-based inflammatory response. The receptor for IL-5 consists of a ligand binding alpha-subunit and a non-ligand binding (common) signal transducing beta-subunit that is shared by the receptors for IL-3 and GM-CSF.
The kinetics of IL-5 binding is still under investigation. Assuming equality with the IL-3 system, (homodimeric) IL-5 binds noncovalently to one IL-5 Rα subunit, which then noncovalently recruits one βc subunit, forming a temporary noncovalently-linked trimer. At this point, a second, newly generated IL-5 R complex can be engaged with the IL-5 Rα subunit from the primary complex by forming a disulfide bond with βc. This is followed by disulfide bonding between the two remaining unlinked receptor components. It is likely that the two βc subunits also become disulphide-linked, creating a functional signal-transducing complex with a stoichiometry of 2:2:2. A newly formed IL-5 R trimer may also link with naturally preformed GM-CSF Rα/βc complexes to form hybrid IL-5 R/GM-CSF R complexes. The function of these complexes and their role with IL-5 activity is currently unknown. As with many cytokines and growth factors, IL-5 has an approximately 15 aa NLS in the body of the molecule. IL-5 with its receptor can be transported into the nucleus following its binding on the cell surface. It is suggested that STATs, which are associated with the receptor, actually enter the receptor via the IL-5 NLS.
Cells known to express IL-5 include eosinophils, NK cells, TC2 CD8+ T cells, mast cells, CD45+ CD4+ T cells, γδ T cells and IL-1β activated endothelial cells. IL-5 is best known for its activity on B cells and eosinophils. Relative to cells, IL-5 appears to induce the differentiation of activated conventional B-2 cells. In mice, IL-5 promotes production of IgA, IgE and IgG1. IL-5 appears to perform a number of functions on eosinophils including growth and differentiation, the down modulation of Mac-1, the upregulation of receptors for IgA and IgG, the stimulation of lipid mediator (leukotriene C4 and PAF) secretion and the induction of granule release. IL-5 may act in an adjunct fashion plays, however, the exact role is unclear. Finally, there is a great deal of interest in the interaction between IL-5 and the CC chemokine eotaxin. Studies suggest that inflammatory-induced and locally produced IL-5 and eotaxin may act on the bone marrow in a cooperative manner.
PRINCIPLE OF THE ASSAY
IL-5 ELISA Kit
IL-5 ELISA Kit is to be used for the in vitro quantitative determination of human interleukin 5 (IL-5) concentrations in serum, plasma, cell culture medium, and urine. This IL-5 ELISA Kit is intended for LABORATORY RESEARCH ONLY and is not to be used for diagnostic or therapeutic purposes. This IL-5 ELISA Kit is a ready-to-use 4.5-hour solid phase immunoassay readily capable of measuring IL-5 levels in serum, plasma, cell culture medium, and urine in the range of 31.25pg/ml to 1000pg/ml. No cross reactivity with other cytokines tested was observed.
This IL-5 ELISA Kitapplies 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-5. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated monoclonal antibody preparation specific for IL-5 and incubated. IL-5 if present, will bind and become immobilized by the antibody pre-coated on the wells and then become “sandwiched” by biotin conjugate. The microtiter plate wells are thoroughly washed to remove unbound IL-5 and other components of the sample. In order to quantitatively determine the amount of IL-5 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, each having 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-5, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.
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