High Sensitive IFN gamma ELISA Assay

Rat IL-6 ELISA Assay

$505.00

The Rat IL-6 ELISA Assay (enzyme-linked immunoassay kit) is intended for the quantitative determination of rat interleukin 6 (IL-6) concentrations in cell culture supernates, serum, and plasma. The Eagle Biosciences Rat Interleukin 6 (IL-6) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

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Rat IL-6 ELISA Assay

The Rat IL-6 ELISA Assay is For Research Use Only

Size: 1×96 wells
Sensitivity: 15 pg/mL
Dynamic Range: 62.5 – 2000 pg/ml
Incubation Time: 3 hours
Sample Type: Serum, Plasma, Cell Culture
Sample Size: 100 µl
Alternative Name: Interleukin 6

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 rat serum or plasma samples are suggested to make a 1:2 dilution.

Assay Principle

The Rat Interleukin 6 (IL-6) ELISA employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Interleukin 6 (IL-6) has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Interleukin 6 (IL-6) 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 6 (IL-6 ) is added to the wells and binds to the combination of capture antibody-Rat interleukin 6 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 6 (IL-6) 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 6 (IL-6) standard dilutions and Interleukin 6 (IL-6) sample concentration determined.

Related Products

Bovine IL-6 ELISA Assay
Mouse IL-6 ELISA Assay

Additional Information

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 and incubate 2 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 (350µL) 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)

Assay Background

Interleukin 6 (IL-6) is multifunctional cytokine that plays important roles in host defense, acute phase reactions, immune responses, nerve cell functions and hematopoiesis (1-5).  IL-6 is a prototypic member of the IL-6 superfamily of cytokines that share gp130 as a component required for signal transduction (4). The rat, mouse and human IL-6 cDNAs have been cloned (6-9). Rat IL-6 cDNA encodes a 211 amino acid (aa) residue precursor polypeptide with a hydrophobic signal peptide that is cleaved to generate the 187 aa residue mature protein. At the protein sequence level, there is approximately 39% identity between rat and human, and 87% identity between mouse and rat IL-6 (6).

IL-6 is expressed by a variety of normal and transformed lymphoid and non-lymphoid cells.  The production of IL-6 is up-regulated by numerous signals such as mitogenic or antigenic stimulation, lipopolysaccharides, calcium ionophores, cytokines and viruses. IL-4, IL-10 and IL-13 inhibit IL-6 expression in monocytes.

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. Hirano, T. (1998) “Interleukin 6” in The Cytokine Handbook, 3rd. ed. Academic Press, New York, p. 197.
2. Hibi, M. et al. (1996) J. Mol. Med. 74:1.
3. Hirano, T. et al. (1994) Stem Cells 12:262.
4. Taga, T. and T. Kishimoto (1997) Annu. Rev. Immunol. 15:797.
5. Van Snick, J. et al. (1990) Annu. Rev. Immunol. 8:253.
6. Northemann, W. et al. (1989) J. Biol. Chem. 264:16072.
7. Van Snick, J. et al. (1988) Eur. J. Immunol. 18:193.
8. Van Snick, J. et al. (1986) Proc. Natl. Acad. Sci. USA 83:9679.
9. Cayphas, S. et al. (1987) J. Immunol. 139:2965.

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