Mouse IL-1 Beta ELISA Assay
The Mouse IL-1 Beta ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 31.25 – 2000 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: Interleukin 1 Beta, IL-1B, IL-1β
SAMPLE COLLECTION AND STORAGE
1. Cell Culture Supernates – Remove particulates by centrifugation.
2. Serum – Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum, avoid hemolysis and high blood lipid samples.
3. Plasma – Recommended EDTA as an anticoagulant in plasma. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection.
4. Assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles.
5. Dilute samples at the appropriate multiple (recommended to do pre-test to determine the dilution factor).
Note: Normal mouse serum or plasma samples are suggested to make a 1:2 dilution.
The Mouse Interleukin 1 Beta (IL-1β) ELISA employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Interleukin 1 Beta (IL-1β) has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Interleukin 1 Beta (IL-1β) present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for Interleukin 1 Beta (IL-1β) is added to the wells and binds to the combination of capture antibody-IL-1β 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 Interleukin 1 Beta (IL-1β) 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 Interleukin 1 Beta (IL-1β) standard dilutions and Interleukin 1 Beta (IL-1β) sample concentration determined.
Mouse IL-1 Alpha ELISA Assay Kit
Mouse IL-2 ELISA Assay
Human IL-1 Beta ELISA Assay
- Prepare all reagents and working standards as directed in the previous sections.
- 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.
- Add 100 µL of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
- 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.
- 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.
- Repeat the aspiration/wash.
- 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.
- Repeat the aspiration/wash.
- Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
- Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- 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)
Interleukin 1 Beta (IL-1β) 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(1-3).
IL-1α and IL-1β are structurally related polypeptides that show approximately 25% homology at the amino acid level (2). Both are synthesized as 31 kDa precursors that are subsequently cleaved into proteins with molecular weights of approximately 17.5 kDa (4, 5). Intracellular IL-1β consists exclusively of the 31 kDa precursor form (6). Extracellular IL-1βconsists of a mixture of both unprocessed and mature IL-1β. These results indicate that processing takes place subsequently to secretion and is not tightly coupled to secretion (7). The specific protease apparently responsible for the processing of IL-1β, designated interleukin-1β-converting enzyme (ICE), has been described (7).
IL-1 possesses a wide variety of biological activities. IL-1 also plays an important role in immune functions, having effects on macrophages/ onocytes, Tlymphocytes, B lymphocytes, NK cells, and LAK cells. It acts on macrophages/ monocytes, inducing its own synthesis as well as the production of TNF and IL-6 (8,9). It activates T cells, resulting in IL-2 production and expression of IL-2 receptors (10). IL-1 also induces the production of GM-CSF and IL-4 from activated T cells (11). It induces B cell proliferation and maturation and increased immunoglobulin synthesis (12, 13).
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.
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. Kurt-Jones, E.A. et al. (1985) Proc. Natl. Acad. Sci. USA 82:1204.
7. Cerretti, D.P. et al. (1992) Science 256:97.USA 86:5943.
8. Levett, D. et al. (1986) J. Immunol. 136:340.
9. Navarro, S. et al. (1989) J. Immunol. 142:4339.
10. Kaye, J. et al. (1984) Lymphokine Res. 3:175.
11. Herrmann, F. et al. (1988) J. Clin. Invest. 81:1415.
12. Abbas, A.K. et al. (1987) Am. J. Pathol. 129:26.
13. Lipsky, P.E. et al. (1983) J. Immunol. 130:2709.