Cardiovascular and Oxidative Stress Assay Kit

Human IL-1a ELISA Assay

$505.00

The Human IL-1a ELISA Assay determines human Interleukin 1 Alpha (IL-1α) concentrations in cell culture supernates, serum, and plasma. The Eagle Biosciences Human Interleukin 1 Alpha (IL-1α) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: L1A31-K01 Categories: , ,

Human IL-1a ELISA Assay

The Human IL-1a ELISA Assay is For Research Use Only

Size: 1×96 wells
Sensitivity: 4 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 1 Alpha, IL-1α, IL-1 Alpha

Assay Background

Interleukin 1 (IL-1) is a name that designates two proteins, IL-1α and IL-1β, which are the products of distinct genes, but which recognize the same cell surface receptors. With the exception of skin keratinocytes, some epithelial cells, and certain cells of the central nervous system, IL-1 is not produced by the cells of healthy individuals. However, in response to stimuli such as those produced by inflammatory agents, infections, or microbial endotoxins, a dramatic increase in the production of IL-1 by macrophages and various other cell types is seen. For reviews on the properties and activities of IL-1α and IL-1β, see references 1 – 3.

IL-1α and IL-1β are structurally related polypeptides that show approximately 25% homology at the amino acid level. Both are synthesized as 31 kDa precursors that are subsequently cleaved into proteins with molecular weights of approximately 17.5 kDa. Neither IL-1α nor IL-1βcontains a typical hydrophobic signal peptide sequence, but evidence suggests that these factors can be secreted by non-classical pathways. A large proportion of IL-1α is retained intracellularly in its precursor form. A portion of this unprocessed IL-1β is transported to the cell surface and remains associated with the cell membrane. The membrane-bound, unprocessed IL-1α is apparently biologically active, acting in a paracrine fashion on adjacent cells having IL-1 receptors. The precursor form of IL-1β, unlike the IL-1α precursor, shows little or no biological activity in comparison to the 17.5 kDa processed form. Intracellular IL-1βconsists exclusively of the 31 kDa precursor form. Extracellular IL-1β consists of a mixture of both unprocessed and mature IL-1β. These results indicate that processing takes place subsequent to secretion and is not tightly coupled to secretion. The specific protease apparently responsible for the processing of IL-1β, designated interleukin 1β-converting enzyme (ICE), has been described.

IL-1α and IL-1β exert their effects by binding to specific receptors. Two distinct receptor types have been isolated that bind both forms of IL-1. An 80 kDa membrane bound receptor protein, IL-1 receptor type I (IL-1 RI), has been isolated from T cells, fibroblasts, keratinocytes, endothelial cells, synovial lining cells, chondrocytes, and hepatocytes. IL-1 RI has been cloned from mouse and human cells and found to be a member of the Ig super family. A second type of IL-1 receptor, IL-1 receptor type II (IL-1 RII), has been found on B cells, neutrophils, and bone marrow cells. This receptor has an apparent molecular weight of about 68 kDa and is also a member of the Ig super family. The two IL-1 receptor types show approximately 28% homology in their extracellular domains, but differ significantly in that the type II receptor has a cytoplasmic domain of only 29 amino acid residues, whereas the type I receptor has a cytoplasmic domain of 213 amino acid residues. In general, IL-1α binds better to the type I receptor and IL-1β binds better to the type II receptor. At present, the mechanisms involved in the transduction of the signal initiated by binding of IL-1 are not well characterized. IL-1 possesses a wide variety of biological activities. It has been shown to induce prostaglandin synthesis in endothelial cells and smooth muscle cells. In the liver, IL-1 initiates the acute phase response resulting in an increase in hepatic protein synthesis and decreased albumin production.

Related Products

Human IL-1 Beta ELISA Assay
Human IL-2 ELISA Assay
Human IL-3 ELISA Kit

Additional Information

Assay Principle

The Eagle Biosciences Human Interleukin 1 Alpha (IL-1α) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-1α has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-1α 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-1α is added to the wells and binds to the combination of capture antibody- IL-1α 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-1α 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-1α standard dilutions and IL-1α sample concentration determined.

  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 100µl 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) 

Documents

Product Manual

 

Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.

Publications

References

1. Dinarello, C.A. (1991) Blood 77:1627.
2. Oppenheim, J.J. et al. (1986) Immunol. Today 7:45.
3. Dinarello, C.A. and S.M. Wolff (1993) New Engl. J. Med. 328:106.
4. Giri, J.G. et al. (1985) J. Immunol. 134:343.
5. Hazuda, D.J. et al. (1988) J. Biol. Chem. 263:8473.
6. Lomedico, P.T. et al. (1984) Nature 312:458.
7. Auron, P.E. et al. (1987) J. Immunol. 138:1447.
8. March, C.J. et al. (1985) Nature 315:641.
9. Rubartelli, A. et al. (1990) EMBO J. 9:1503.
10. Rubartelli, A. et al. (1993) Cytokine 5:117.
11. Kurt-Jones, E.A. et al. (1985) Proc. Natl. Acad. Sci. USA 82:1204.
12. Hazuda, D. et al. (1989) J. Biol. Chem. 264:1689.
13. Hazuda, D. et al. (1990) J. Biol. Chem. 265:6318.
14. Cerretti, D.P. et al. (1992) Science 256:97.
15. Urdal, D.L. et al. (1988) J. Biol. Chem. 263:2870.
16. Sims, J.E. et al. (1988) Science 241:585.
17. Dinarello, C.A. (1988) Blood Purif. 6:164.
18. Dejuna, E. et al. (1987) Blood 69:635.
19. Ramadori, G. et al. (1985) J. Exp. Med. 162:930.

Product Citations