IL-5 Human ELISA Assay

$430.00

The Human IL-5 ELISA Assay (enzyme-linked immunoassay kit) is intended for the quantitative determination of human Interleukin 5 (IL-5) concentrations in cell culture supernates, serum, and plasma. The Human Interleukin 5 (IL-5) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: IL531-K01 Categories: , ,

IL-5 Human ELISA Assay

The IL-5 Human ELISA Assay is For Research Use Only

Size: 1×96 wells
Sensitivity: 2 pg/mL
Dynamic Range: 7.8 – 250 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: Interleukin 5


Assay Background

Interleukin-5 (IL-5) is a secreted glycoprotein that belongs to the α-helical group of cytokines. Unlike other family members, it is present as a covalently linked antiparallel dimer. The cDNA for human IL-5 encodes a signal peptide and a 115 amino acid (aa) mature protein. Mature human IL-5 shares 70%, 70%, 62%, 71%, 70% and 66%, aa sequence identity with mouse, rat, canine, equine, feline and porcine IL-5, respectively and shows cross-reactivity with mouse IL-5. IL-5 is primarily produced by CD4+ Th2 cells, but also by activated eosinophils, mast cells, EBV-transformed B cells, Reed-Sternberg cells in Hodgkin’s disease, and IL-2-stimulated invariant natural killer T cells (iNKT). IL-5 increases production and mobilization of eosinophils and CD34+ progenitors from the bone marrow and causes maturation of eosinophil precursors outside the bone marrow. The receptor for human IL-5, mainly expressed by eosinophils, but also found on basophils and mast cells, consists of a unique ligand-binding subunit (IL-5 Rα) and a shared Signal transducing subunit, βc. IL-5 Rα first binds IL-5 at low affinity, then associates with preformed βc dimers, forming a high-affinity receptor (12). IL-5 also binds proteoglycans, potentially enhancing its activity. Soluble forms of IL-5 Rα antagonize IL-5 and can be found in vivo. In humans, IL-5 primarily affects cells of the eosinophilic lineage, and promotes their differentiation, maturation, activation, migration and survival, while in mice IL-5 also enhances Ig class switching and release from B1 cells. IL-5 also promotes differentiation of basophils and primes them for histamine and leukotriene release.


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Additional Information

Assay Principle


The Human Interleukin 5 (IL-5) ELISA employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-5 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-5 present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for IL-5 is added to the wells and binds to the combination of capture antibody- IL-5 in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A colored product is formed in proportion to the amount of IL-5 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven IL-5 standard dilutions and IL-5 sample concentration determined.

Assay Procedure


  1. Prepare all reagents and working standards as directed in the previous sections.
  2. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
  3. Add 100 µl of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
  4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µl) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
  5. Add 100 µl of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
  6. Repeat the aspiration/wash.
  7. Add 100 µl of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C Avoid placing the plate in direct light.
  8. Repeat the aspiration/wash.
  9. Add 100 µl of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
  10. Add 100 µl of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  11. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length;610-650nm is acceptable) 

Manual

Product Manual


Publications

References


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