Human IL-32a ELISA Assay
The Human IL-32a ELISA Assay is For Research Use Only
Size: 1×96 wells
Sensitivity: 7 pg/mL
Dynamic Range: 15.625 – 500 pg/ml
Incubation Time: 3.5 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Names: IL-32 Alpha, Interleukin 32 alpha, IL-32α
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 human serum or plasma samples are suggested to make a 1:2 dilution.
IL-32α is the shortest and most abundant of four potential splice variants of the proinflammatory cytokine IL-32 (previously called NK4) with a predicted unmodified size of 15 kDa. Potential modifications include myristoylation and N-glycosylation. The IL-32α shows increased potency at inducing CXCL2/MIP-2 and CXCL8 expression in PBMC relative to uncleaved IL-32α. Transfected IL-32α was more likely to be cell associated as compared to IL-32β, suggesting an intracellular function. IL-32 is induced by mitogens in peripheral lymphocytes, by IFNγ in epithelial cells, or by IL-12 with IL-18 in NK cells and in turn induces cytokine expression. These activities result in epidermal hyperplasia in models of human skin.
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Human IL-21 ELISA Assay
The Eagle Biosciences Human Interleukin 32 Alpha (IL-32α) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-32α Α has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-32α 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-32α is added to the wells and binds to the combination of capture antibody- IL-32α 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-32α 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-32α standard dilutions and IL-32α sample concentration determined.
- 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 (350mL) 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)
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. Dahl, C. A.et al. (1992) J Immunol. 148: 597.
2. Kim, S. H. et al. (2005) Immunity. 22: 131.
3. Goda, C. et al. (2006)Int Immunol. 18:233.
4. Dinarello, C. A. et al. (2006)Ann Rheum Dis. 65: 61.
5. Novick, D. et al. (2006)Proc Natl Acad Sci U S A.103:3316.
6. Netea, M. G. et al. (2008) Proc Natl Acad Sci U S A. 105: 3515.
7. Joosten, L. A. et al. (2006)Proc Natl Acad Sci U S A. 103: 3298.
8. Nold, M. F. et al. (2008)J Immunol.181: 557.
9. Rasool, S. T. et al. (2008)Immunol Lett.117: 161.
10. Netea, M. G. et al. (2006)PLoS Med. 3:e277.