Human IL-9 ELISA Assay Kit

$405.00

The Eagle Biosciences Human Interleukin 9 (IL-9) ELISA Assay Kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of human Interleukin 9 (IL-9) concentrations in cell culture supernates, serum, and plasma. The Eagle Biosciences Human Interleukin 9 (IL-9) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: IL931-K01 Categories: , ,

Human IL-9 ELISA Assay Kit

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

Additional Information

Assay Background


Human IL-9 was originally identified as a cytokine found in the conditioned medium of a human T cell leukemia virus type I (HTLV-I) transformed T cell line that is mitogenic for the factor-dependent human megakaryoblastic leukemic cell line, M07e. This human cytokine and its murine homologue are now designated as human and mouse IL-9. The gene for H IL-9 has been mapped to human chromosome 5. As in the mouse system, the human IL-9 cDNA encodes a 144 amino acid residue precursor protein with an 18 amino acid signal peptide that is cleaved to form the mature cysteine rich protein with a predicted molecular mass of 14 kDa. Human IL-9 contains four potential Nlinked glycosylation sites and the native HIL-9 is a highly glycosylated protein. Human and mouse IL-9 share 56% and 67% homology at the amino acid and nucleotide levels, respectively. Although murine IL-9 is active on human cells, human IL-9 is not active on mouse cells. Human and murine IL9 are also capable of enhancing in vitro survival of human T cell lines as well as synergizing with Epo to support erythroid colony formation in vitro.

Assay Principle


The Eagle Biosciences Human Interleukin 9 (IL-9) ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-9 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-9 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-9 is added to the wells and binds to the combination of capture antibody- IL-9 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-9 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-9 standard dilutions and IL-9 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 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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4.    Yin, T. et al. (1995) J. Biol. Chem. 270: 20497..
5.    Nicolaides, N.C. et al. (1998). Proc. Natl. Acad. Sci. U.S.A. 94: 13175
6.    Little, F.F. et al. (2001) Am. J. Respir. Cell Mol. Biol. 25 : 347.